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• Research Paper Volume 14, Issue 2 pp 678-707

  Targeting regulation of ATP synthase 5 alpha/beta dimerization alleviates senescence

  Relevance score: 6.365692

  Yun Haeng Lee, Doyoung Choi, Geonhee Jang, Ji Yun Park, Eun Seon Song, Haneur Lee, Myeong Uk Kuk, Junghyun Joo, Soon Kil Ahn, Youngjoo Byun, Joon Tae Park

  Keywords: senescence amelioration, KB1541, 14–3–3ζ, ATPase synthase 5, OXPHOS

  Published in Aging on January 30, 2022

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  Senescence is a distinct set of changes in the senescence-associated secretory phenotype (SASP) and leads to aging and age-related diseases. Here, we screened compounds that could ameliorate senescence and identified an oxazoloquinoline analog (KB1541) designed to inhibit IL-33 signaling pathway. To elucidate the mechanism of action of KB1541, the proteins binding to KB1541 were investigated, and an interaction between KB1541 and 14–3–3ζ protein was found. Specifically, KB1541 interacted with 14–3–3ζ protein and phosphorylated of 14–3–3ζ protein at serine 58 residue. This phosphorylation increased ATP synthase 5 alpha/beta dimerization, which in turn promoted ATP production through increased oxidative phosphorylation (OXPHOS) efficiency. Then, the increased OXPHOS efficiency induced the recovery of mitochondrial function, coupled with senescence alleviation. Taken together, our results demonstrate a mechanism by which senescence is regulated by ATP synthase 5 alpha/beta dimerization upon fine-tuning of KB1541-mediated 14–3–3ζ protein activity.

• Research Paper Volume 10, Issue 8 pp 1867-1883

  Matcha green tea (MGT) inhibits the propagation of cancer stem cells (CSCs), by targeting mitochondrial metabolism, glycolysis and multiple cell signalling pathways

  Relevance score: 5.6793113

  Gloria Bonuccelli, Federica Sotgia, Michael P. Lisanti

  Keywords: Matcha green tea, cancer stem-like cells (CSCs), proteomics analysis, metabolism, mitochondrial OXPHOS, glycolysis

  Published in Aging on August 23, 2018

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  Matcha green tea (MGT) is a natural product that is currently used as a dietary supplement and may have significant anti-cancer properties. However, the molecular mechanism(s) underpinning its potential health benefits remain largely unknown. Here, we used MCF7 cells (an ER(+) human breast cancer cell line) as a model system, to systematically dissect the effects of MGT at the cellular level, via i) metabolic phenotyping and ii) unbiased proteomics analysis. Our results indicate that MGT is indeed sufficient to inhibit the propagation of breast cancer stem cells (CSCs), with an IC-50 of ~0.2 mg/ml, in tissue culture. Interestingly, metabolic phenotyping revealed that treatment with MGT is sufficient to suppress both oxidative mitochondrial metabolism (OXPHOS) and glycolytic flux, shifting cancer cells towards a more quiescent metabolic state. Unbiased label-free proteomics analysis identified the specific mitochondrial proteins and glycolytic enzymes that were down-regulated by MGT treatment. Moreover, to discover the underlying signalling pathways involved in this metabolic shift, we subjected our proteomics data sets to bio-informatics interrogation via Ingenuity Pathway Analysis (IPA) software. Our results indicate that MGT strongly affected mTOR signalling, specifically down-regulating many components of the 40S ribosome. This raises the intriguing possibility that MGT can be used as inhibitor of mTOR, instead of chemical compounds, such as rapamycin. In addition, other key pathways were affected, including the anti-oxidant response, cell cycle regulation, as well as interleukin signalling. Our results are consistent with the idea that MGT may have significant therapeutic potential, by mediating the metabolic reprogramming of cancer cells.

  

  MGT treatment reduces stemness in MCF7 breast cancer cells. (A) The effects of 0.2 mg/ml MGT on cell proliferation were tested on MCF7 cells in monolayer by SRB assay. Note that MGT only slightly reduced viability of bulk cancer cells by 8%. (B) Importantly, MGT inhibited the sphere-forming ability of MCF7 cells by 50%. Bar graphs are shown as the mean ± SEM; t-test, two-tailed test. **p < 0.005, ***p < 0.0001.

  
  
  

  

  MGT treatment reduces basal respiration and ATP production in MCF7 cells. MCF7 cells were seeded at the density of six thousands cells in 96-wells plates. After twenty-four hours, filtered 0.2 mg/ml MGT was added and incubated for seventy-two hours. Oxygen consumption rate (OCR) was measured by Seahorse XF Analyser. Top panel: representative trace; bottom panel: bar graph with OCR quantification. Note that MGT treatment significantly decreases the basal respiration and the ATP production as compared to control cells. Others parameters, such as proton leak, maximal and spare respirations did not significantly change. Experiments were performed 3 times, with six repeats for each replicate. Bar graphs are shown as the mean ± SEM; t-test, two-tailed test. ***p < 0.0001.

  
  
  

  

  MGT treatment inhibits glycolysis of MCF7 cells. MCF7 cells were seeded and treated with MGT as described above. Extracellular consumption rate (ECAR) was assessed by Seahorse XF Analyser. Top panel: representative trace; bottom panel: bar graph with OCR quantification. Importantly, the treatment significantly reduced the glycolysis and the glycolytic capacity as compared to control cells. Experiments were performed in triplicate, six repeats for each replicate. Bar graphs are shown as the mean ± SEM, t-test, two-tailed test. *p < 0.05.

  
  
  

  

  Table 1A. Commonly down-regulated proteins in MCF7 cells after treatment with doxycycline or MGT. List of down-regulated mitochondrial proteins and relative fold change.

  
  
  

  

  Table 1B. Commonly down-regulated proteins in MCF7 cells after treatment with doxycycline or MGT. List of down-regulated glycolytic proteins and relative fold change.

  
  
  

  

  Venn diagram of proteomics data of MGT-treated MCF7 cells versus doxycycline-treated MCF7 cells. Proteomic analysis validates the metabolic effects of MGT on breast cancer cells. (A) Venn diagram of mitochondrial down-regulated proteins in MGT-treated cells versus doxycycline-treated cells. Note that, among the mitochondrial down-regulated proteins by the two treatments, eight are commonly down-regulated. (B) Venn diagram of glycolytic down-regulated proteins in MGT-treated cells versus doxycycline-treated cells. Note that the two different treatments down-regulated several glycolytic proteins and four of those were in common among the two.

  
  
  

  

  Canonical pathways affected by MGT in MCF7 cells. Ingenuity pathways analysis (IPA) showed the cellular pathways most significantly (p<0.05) affected by MGT treatment. The p value for each pathway is represented with a bar and reported as the negative log of the p value.

  
  
  

  

  IPA analysis: Schematic representation of mTOR pathway. IPA analysis revealed changes in the expression of proteins involved in mTOR signaling after MGT treatment for 48 hours. In this map, the 40S ribosome was indicated as dramatically down-regulated (intense green color), suggesting likely inhibition of protein translation.

  
  
  

  

  Impairment of mitochondrial functions uncovered by IPA analysis. Depicted is the map of oxidative phosphorylation. All the mitochondrial complexes are affected by the treatment, particularly complex III is dramatically down-regulated, as indicated by the intense green colour.

  
  
  

  

  Modifications in the expression of glycolytic enzymes. Schematic representation of glucose metabolism upon MGT treatment. Green tea exposure causes an evident impairment of glycolytic pathway. Proteins down-regulated (in green) or up-regulated (in red) are shown.

  
  
  

  

  Changes in the expression of proteins implicated in the pentose phosphate pathway. MGT treatment up-regulates the expression of proteins involved in the PPP pathway (red color).

  
  
  

  

  MGT treatment affects cell cycle regulation in MCF7 cells. Schematic representation of cell cycle regulation upon treatment with green tea. Proteins down-regulated (in green) or up-regulated (in red) are shown.

  
  
  

  

  Overview of the metabolic and cellular processes affected by MGT. Note that MGT treatment inhibits cellular metabolism (glycolysis, mitochondrial respiration, fatty acid synthesis) with a likely compensatory increase in fatty acid breakdown.

  
  
  

• Research Paper Volume 9, Issue 12 pp 2610-2628

  Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy

  Relevance score: 5.3178945

  Bela Ozsvari, Gloria Bonuccelli, Rosa Sanchez-Alvarez, Richard Foster, Federica Sotgia, Michael P. Lisanti

  Keywords: Vitamin B2 (Riboflavin), DPI (Diphenyleneiodonium chloride), mitochondria, Mitoflavoscin, flavin enzymes, OXPHOS, cancer stem-like cells (CSCs)

  Published in Aging on December 16, 2017

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  Here, we performed high-throughput drug-screening to identify new non-toxic mitochondrial inhibitors. This screening platform was specifically designed to detect compounds that selectively deplete cellular ATP levels, but have little or no toxic side effects on cell viability. Using this approach, we identified DPI (Diphenyleneiodonium chloride) as a new potential therapeutic agent. Mechanistically, DPI potently blocks mitochondrial respiration by inhibiting flavin-containing enzymes (FMN and FAD-dependent), which form part of Complex I and II. Interestingly, DPI induced a chemo-quiescence phenotype that potently inhibited the propagation of CSCs, with an IC-50 of 3.2 nano-molar. Virtually identical results were obtained using CSC markers, such as CD44 and CD24. We further validated the effects of DPI on cellular metabolism. At 10 nM, DPI inhibited oxidative mitochondrial metabolism (OXPHOS), reducing mitochondrial driven ATP production by >90%. This resulted in a purely glycolytic phenotype, with elevated L-lactate production. We show that this metabolic inflexibility could be rapidly-induced, after only 1 hour of DPI treatment. Remarkably, the mitochondrial inhibitory effects of DPI were reversible, and DPI did not induce ROS production. Cells maintained in DPI for 1 month showed little or no mitochondrial activity, but remained viable. Thus, it appears that DPI behaves as a new type of mitochondrial inhibitor, which maintains cells in a state of metabolic-quiescence or “suspended animation”.In conclusion, DPI treatment can be used to acutely confer a mitochondrial-deficient phenotype, which we show effectively depletes CSCs from the heterogeneous cancer cell population. These findings have significant therapeutic implications for potently targeting CSCs, while minimizing toxic side effects. We also discuss the possible implications of DPI for the aging process. Interestingly, previous studies in C. elegans have shown that DPI prevents the accumulation of lipofuscin (an aging-associated hallmark), during the response to oxidative stress. Our current results are consistent with data showing that flavins (FAD, FMN and/or Riboflavin) are auto-fluorescent markers of i) increased mitochondrial “power” (OXPHOS) and ii) elevated CSC activity.Finally, we believe that DPI is one of the most potent and highly selective CSC inhibitors discovered to date. Therefore, our current findings suggest a new impetus to create novel analogues of i) DPI (Diphenyleneiodonium chloride) and ii) DPI-related compounds (Diphenyliodonium chloride), using medicinal chemistry, to optimize this very promising and potent anti-CSC activity. We propose to call these new molecules “Mitoflavoscins”.For example, DPI is ~30 times more potent than Palbociclib (IC-50 = 100 nM), which is an FDA-approved CDK4/6 inhibitor, that broadly targets proliferation in any cell type, including CSCs.

  

  Diagram illustrating the main steps of our drug discovery work-flow. (i) Phenotypic drug screening. A sub-set of the Tocriscreen compound library was subjected to phenotypic drug screening, at a concentration of 20 µM. The screen was set up to specifically identify compounds which can functionally induce ATP-depletion, without inducing cell death. Subsequently, positive hits were re-screened at a lower concentration (10 µM). (ii) Functional validation. Hit compounds were then further validated using mammosphere assays (for assessing potential anti-cancer stem cell activity). Metabolic flux analysis (to determine specific effects on oxygen consumption), flow cytometry and viability assays were also carried out. (iii) Top hit compound. The structure of DPI (Diphenyleneiodonium chloride), the top hit compound, is shown. Importantly, DPI is known to functionally target flavin-containing enzymes, especially within mitochondrial complex I (NDUFV1/2/3) and II (SDHA), as well as the TCA cycle. DPI chemically reacts with and inactivates FMN (flavin mononucleotide).

  
  
  

  

  DPI selectively depletes ATP without inducing massive cell death. MCF7 cells were treated with DPI for 72 hours and were first subjected to fluorescent Hoechst staining (DNA content) and then to the luminescent measurement of the ATP content in the same wells, using CellTiter-Glo as a probe. Note that at 72 hours, 500 nM DPI selectively depletes ATP levels by >80%, but does not significantly induce cell death, as the number of cells attached to the plate remains the unchanged (as detected by DNA content).

  
  
  

  

  DPI does not significantly affect cell viability, even after 5 days of treatment. Cell viability was determined by employing the Sulphorhodamine B (SRB) assay, to measure total protein content. Note that after 5 days of incubation, DPI shows little or no toxicity in MCF7 cells, at a concentration as high as 33 nM. However, some toxicity was observed at 100 nM. Similarly, normal fibroblasts (hTERT-BJ1) showed little or no toxic effects at up to 100 nM, after 5 days of incubation.

  
  
  

  

  DPI potently inhibits mitochondrial respiration. After 24 hours of treatment with DPI (2.5 to 50 nM), MCF7 cells were subjected to metabolic flux analysis with the Seahorse XFe96, which measures the OCR (the oxygen consumption rate). Note that at concentration of 2.5 nM, little or no effect was observed. However, at 5 nM, basal respiration was reduced by ~ 50%. *** p<0.001.

  
  
  

  

  DPI induces a reactive glycolytic response. After 24 hours of treatment with DPI (2.5 to 50 nM), MCF7 cells were subjected to metabolic flux analysis with the Seahorse XFe96, which also measures ECAR (the extracellular acidification rate), a surrogate marker for L-lactate production. Note that at a concentration of 2.5 nM, little or no effect was observed. However, at 10 nM, glycolysis was increased by 2-fold. *** p<0.001.

  
  
  

  

  DPI drives the production of L-lactate. After treatment with DPI (5 or 50 nM) for 1, 3 or 5 days, the cell culture media from MCF7 cells was subjected to analysis using the ISCUS-flex microdialysis analyser, to directly measure L-lactate content. Note that DPI induces significant L-lactate production, nearly doubling the amount of lactate as early as 1 day of treatment, using only 5 nM DPI. *** p<0.001.

  
  
  

  

  Dose-dependent inhibition of CSC propagation using DPI, as measured using the mammosphere assay. MCF7 cells were seeded into low-attachment plates and allowed to form mammospheres (a.k.a., 3D tumor spheres), over a period of 5 days. Note that DPI markedly reduced CSC propagation, with an IC-50 of 3.23 nM. The mammosphere assay was performed over the range of 0.2 nM to 10 µM.

  
  
  

  

  DPI selectively eliminates CSCs from the total cell population. MCF7 cells were cultured for 5 days as monolayers, in the presence of DPI (5, 10 and 50 nM). Then, the cells were harvested and subjected to FACS analysis to determine the levels of CSC markers. Panel (A) shows that the CD44+/CD24- cell population (which serves as a marker for breast CSCs) is dose-dependently reduced by DPI treatment, with an IC-50 of 10 nM. Panel (B) contains dot plots showing the double fluorescent CD44+/CD24- FACS assay. Note that the signal has significantly decreased in the lower right quadrant (Q1), after DPI treatment. * p<0.05, ** p<0.01.

  
  
  

  

  Effect of DPI treatment on mitochondrial ROS production.The effects of DPI on mitochondrial ROS production were determined over the range of 5 to 50 nM. Note that at a concentration of 5 nM, DPI failed to induce mitochondrial ROS production. In contrast, 50 nM DPI induced the same amount of mitochondrial ROS as 500 nM Rotenone, which was used as a positive control. Mitochondrial ROS production was monitored by FACS analysis, using MitoSOX as a fluorescent indicator. * p<0.05.

  
  
  

  

  DPI is generally “non-toxic” and does not increase the apoptotic rate in MCF7 cell monolayer cultures. Briefly, 300,000 MCF7 cells were plated in 6-well plates in complete media supplemented with 10% HiFBS. On the next day, the cells were treated with DPI (5, 10, or 50 nM) for 24 hours. Vehicle alone (DMSO) for control cells were processed in parallel. At least 30,000 events were recorded by FACS using LSRII. The results presented are the average of three biological replicates analyzed in independent experiments and are expressed as mean fluorescence intensity. (A) Bar-graphs are used to summarize the overall results; (B) Representative FACS tracings are also shown. Note that DPI fails to significantly increase the apoptotic rate in MCF7 cell monolayers.

  
  
  

  

  DPI rapidly induces the inhibition of mitochondrial respiration. Even with as little as 1 hour of DPI treatment, the mitochondrial oxygen consumption rate (OCR) was progressively reduced, over a concentration range of 10 to 100 nM, as seen using the Seahorse XFe96 Metabolic Flux Analyzer. Note that basal respiration was inhibited with an IC-50 of 50 nM.

  
  
  

  

  DPI rapidly induces a reactive glycolytic phenotype. Even with as little as 1 hour of DPI treatment, glycolysis was progressively increased, over a concentration range of 5 to 100 nM, as seen using the Seahorse XFe96 Metabolic Flux Analyzer. Note that glycolysis was effectively doubled.

  
  
  

  

  The inhibitory effects of DPI on mitochondrial respiration are reversible. To assess the reversibility of DPI’s effects after drug removal, MCF7 cells were first subjected to DPI treatment for 24 hours. Then, the DPI was removed by washing with normal media and the cells were cultured for an additional 24 hours to allow them to recover. This cycle of DPI-treatment, “wash-out” and recovery was carried out over a concentration range of 10 to 50 nM DPI. Note that with 10 nM DPI, there was a near complete recovery of basal respiration, after only 24 hours. Higher concentrations (25 and 50 nM) still showed significant recovery, but the recovery was not complete at this time point.

  
  
  

  

  The inhibitory effects of DPI on mitochondrial respiration are reversible: Focus on 10 nM. As in Figure 12, except that Panel C is shown instead as a series of bar graphs, to better illustrate and separate the different metabolic parameters.

  
  
  

  

  Long-term treatment with DPI is surprisingly “non-toxic”. To determine the long-term effects of DPI (10, 25 and 50 nM), MCF7 cells were cultured for an entire month, in the presence of the drug. Then, their mitochondrial respiration was assessed by metabolic flux analysis. Panel (A) shows that all three drug concentrations show near complete inhibition of mitochondrial respiration. Panel (B) illustrates the morphology of cells after 4 weeks of DPI treatment. Note that the morphology and density of the cells is relatively unchanged, especially at a DPI concentration of 10 nM. Media with DPI or vehicle alone was replaced every 2 to 3 days, during the period of 1 month. Cells undergoing long-term treatment with DPI were also successfully passaged, after harvesting by standard trypsinization techniques.

  
  
  

  

  The chemical structures of (A) DPI and (B) FMN are compared. It has been proposed that the effects of DPI are mediated through the general inhibition of flavo-enzymes, such as mitochondrial Complex I (NADH dehydrogenase), via the targeting of FMN. The three known flavin-containing protein components of Complex I are: NDUFV1 (51 kD), NDUFV2 (24 kD) and NDUFV3 (10 kD). It has been suggested that DPI chemically reacts with FMN, interrupting its function and impairing electron transport. In the human genome, there are ~90 flavo-proteins; more than two-thirds require FAD, while only ~15% require FMN. Flavo-proteins are very often localized to the mitochondria, because of their role in redox reactions. Nearly all flavo-proteins (~90%) catalyze some form of redox reaction.

  
  
  

  

  Targeting flavin-containing enzymes eradicates CSCs. Flavins (FMN, FAD and Riboflavin) have been independently used as markers for high mitochondrial OXPHOS or increased CSC activity. However, it remained unknown whether flavins were selectively required for CSC propagation. Here, we showed that DPI, which is known to specifically target flavin-containing enzymes, behaves as a powerful mitochondrial OXPHOS inhibitor and successfully eradicates CSCs with high potency, in the low nano-molar range. Therefore, these findings provide the first proof-of-concept that inhibiting flavin-containing enzymes is a new viable strategy for effectively targeting CSCs.

  
  
  

  

  Comparison of the structures of (A) Diphenyleneiodonium (DPI), with the related compound (B) Diphenyliodonium chloride. Note the key similarities between these two chemical structures. Both of these classes of molecules target flavin-containing proteins.

  
  
  

• Research Paper Volume 2, Issue 11 pp 843-853

  Activation of mitochondrial energy metabolism protects against cardiac failure

  Relevance score: 7.7270274

  Tim J. Schulz, Dirk Westermann, Frank Isken, Anja Voigt, Beate Laube, René Thierbach, Doreen Kuhlow, Kim Zarse, Lutz Schomburg, Andreas F. H. Pfeiffer, Carsten Tschöpe, Michael Ristow

  Keywords: OXPHOS, cardiac failure, cardiomyopathy, insulin signaling, mitohormesis

  Published in Aging on November 16, 2010

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  Cardiac failure is the most prevalent cause of death at higher age, and is commonly associated with impaired energy homeostasis in the heart. Mitochondrial metabolism appears critical to sustain cardiac function to counteract aging. In this study, we generated mice transgenically over-expressing the mitochondrial protein frataxin, which promotes mitochondrial energy conversion by controlling iron-sulfur-cluster biogenesis and hereby mitochondrial electron flux. Hearts of transgenic mice displayed increased mitochondrial energy metabolism and induced stress defense mechanisms, while overall oxidative stress was decreased. Following standardized exposure to doxorubicin to induce experimental cardiomyopathy, cardiac function and survival was significantly improved in the transgenic mice. The insulin/IGF-1 signaling cascade is an important pathway that regulates survival following cytotoxic stress through the downstream targets protein kinase B, Akt, and glycogen synthase kinase 3. Activation of this cascade is markedly inhibited in the hearts of wild-type mice following induction of cardiomyopathy. By contrast, transgenic overexpression of frataxin rescues impaired insulin/IGF-1 signaling and provides a mechanism to explain enhanced cardiac stress resistance in transgenic mice. Taken together, these findings suggest that increased mitochondrial metabolism elicits an adaptive response due to mildly increased oxidative stress as a consequence of increased oxidative energy conversion, previously named mitohormesis. This in turn activates protective mechanisms which counteract cardiotoxic stress and promote survival in states of experimental cardiomyopathy. Thus, induction of mitochondrial metabolism may be considered part of a generally protective mechanism to prevent cardiomyopathy and cardiac failure.

  

  (A) Representative anti-hemagglutinin immunoblot showing several tissues from a transgene-negative littermate (“-”) and a transgenic (“+”) animal each. “Control” is a previously published cell line over-expressing frataxin [20]. (B) Aconitase activity measured in murine heart samples. Grey bars indicate wild-type (WT) and white bars indicate frataxin-transgenic (FX) animals (also applies to subsequent figures). (C) ATP, (D) NADH, (E)) NADPH, (F) reduced glutathione (GSH) and (G) thiobarbituric acid reactive substances (TBARS) contents in the hearts of wild-type and frataxin-transgenic animals. Error bars represent S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001 (applies to this and all subsequent figures) (n=4).

  
  
  

  

  (A) Maximum rate of pressure development in the left ventricle (dP/dtmax), (B) left-ventricular maximum rate of pressure decrease (dP/dtmin), (C) end-systolic pressure (Pes), (D) end-systolic volume (Ves), (E) end-diastolic pressure (Ped), (F) end-diastolic volume (Ved), (G) time constant of isovolumetric left ventricular pressure decline (tau, τ). (H) stroke work, (I) stroke volume, (J) cardiac output, (K) ejection fraction, and (L) heart rate in wild-type vs. frataxin-transgenic animals following administration of doxorubicin. Grey bars depict wild-type litter mates, white bars frataxin-transgenic mice (n=6 each).

  
  
  

  

  Survival plot of animals exposed to doxorubicin. Triangles depict frataxin-transgenic (FX) animals; squares depict wild-type (WT) animals (n=24 per genotype).

  
  
  

  

  Three animals were analyzed by western blotting for each group. Lanes 1-3: WT untreated; lanes 4-6: FX untreated; lanes 7-9: WT doxorubicin-treated; lanes 10-12: FX doxorubicin-treated. Membranes were probed with the indicated antibody, then stripped and re-probed with α-tubulin as loading control. Approximate protein band size to the left as indicated by arrows.