Research Paper Volume 14, Issue 7 pp 2966—2988

Figure 4. Effects of serial passage and IGFBP5 knockdown on Akt and ERK phosphorylation in MEFs. (A) Representative immunoblots for phospho-Ser473-Akt (pAkt), Akt, phospho-Ser9-GSK3β (pGSK3β), GSK3β, and β-actin in P2 and P8 MEFs. (B) Quantitative data for pAkt and pGSK3β levels normalized to level of the corresponding total protein. N=14 from four independent experiments. *P<0.05, **P<0.01 by unpaired Student’s t-test. (C) Representative immunoblots for pAkt, Akt, pGSK3β and GSK3β in P2 MEFs transfected with control and Igfbp5 siRNA. (D) Quantitative data for pAkt and pGSK3β levels normalized to level of the corresponding total protein. N=6. (E) Representative immunoblots for phospho-Thr202/Tyr204-ERK1/2 (pERK1 and pERK2), ERK1, ERK2, and β-actin in P2, P4, P6, and P8 MEFs. (F) Quantitative data for pERK1, pERK2, total ERK1, and total ERK2. N=12 in each passage from five independent experiments. *P<0.05, **P<0.01, ***P<0.001 by one-way repeated measures ANOVA with a Student-Newman-Keuls test. (G) Representative immunoblots for pERK1/2, total ERK1/2, and β-actin in P2 MEFs transfected with control or Igfbp5 siRNA. (H) Quantitative data for pERK1, pERK2, total ERK1, and total ERK2 in cells transfected with control or Igfbp5 siRNA. N=11 in each treatment from six independent experiments. ***P<0.001 by unpaired Student’s t-test. (I) Representative immunoblots for phospho-Ser217/221-MEK1/2 (pMEK1/2), total MEK1/2, and β-actin in P2 MEFs transfected with control or Igfbp5 siRNA. (J) Quantitative data for pMEK1/2 and MEK1/2 in cells transfected with control or Igfbp5 siRNA. N=7 in each treatment from seven independent experiments. *P<0.05 by paired Student’s t-test. Data are represented as mean +/- SEM. kDa: kilodalton. a.u.: arbitrary unit. NS: not significant.