Research Paper Volume 14, Issue 21 pp 8745—8762

Figure 3. Golph3 regulates GA stress by interacting with vimentin. GST-Golph3 protein was expressed and purified from the Escherichia coli expression system, and cell lysates were precipitated with glutathione Sepharose beads and immunoblotted by SDS-PAGE, the obviously precipitated proteins in the SDS gel were tested by mass spectrometry (A) and immunoblotted with Vimentin antibody (B). (C) Lysates from BV2 microglial cells were immunoprecipitated with a Golph3 antibody or rabbit IgG and immunoblotted with a mouse vimentin antibody. (D) The colocalization of NLRP3 and VPS35 was tested by immunofluorescence method. Bar = 2 μm. (E) Western blot analysis of Vimentin in WT, Golph3 KO BV2 cells treated with or without HG. GAPDH was used as an internal control for normalization. (F) Western blot analysis of Golph3 in WT, Vimentin KO BV2 cells treated with or without HG. GAPDH was used as an internal control for normalization. (G) Immunofluorescence detection of GM130 in WT and Vimentin KO BV2 cells, the percent of cells with dispersed Golgi apparatus normalized with total cells. Bar = 2 μm. Data represent means ± SEM of 3 independent experiments. * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 according to two-way ANOVA with Bonferroni's post hoc test.