Figure 3
EtOH-induced Golgi fragmentation is regulated by Rab6a GTPase.
(a,b) Immunostaining of Rab6a (green) and giantin (red) in (a) VA-13 cells: control, EtOH- or SAR1A siRNA-treated; (b) immunostaining of giantin (green) and Rab6a (red) in hepatocytes isolated from control and EtOH-fed rats. The right panels show green and red channels corresponding to Golgi and cytoplasmic regions (white boxes). Nuclei were counterstained with DAPI (blue). All confocal images were acquired with same imaging parameters; bars, 10 μm. (c) Quantification summarizing the Rab6a-specific fluorescence signal colocalized with giantin in cells presented in (a,b). The Pearson’s coefficient is presented as a mean ± SD from three independent experiments; *p < 0.001. (d) Immunostaining of giantin in VA-13 cells transiently transfected with empty PCMV vector (Ctrl), treated with 35 mM EtOH for 72 h and simultaneously transiently transfected with empty PCMV vector (EtOH), or transfected with Rab6(T27N) or NMIIAΔtailpiece plasmids. (e) Quantification of the fragmented Golgi in cells treated as described in the (d) n = 60 cells from three independent experiments. (f) NMIIA-P-S1943 Western blot of the Golgi fraction isolated from hepatocytes of control and EtOH-fed rats. Golgi membranes were isolated from cells as described in Methods and normalized by the total protein. EtOH sample was treated with CIP in the presence or absence of β-GP. (g,h) NMIIA-P-S1943 and Rab6a (g) and giantin and Rab6a (h) Western blot of the complexes pulled down with Rab6a Ab from the lysate of control VA-13 cells or cells treated with 35 mM EtOH for 72 h. Input was normalized by NMIIA-P-S1943 (g) or giantin (h). (i) Giantin Western blot of the lysates of VA-13 cells treated with scramble or GOLGB1 (giantin) siRNAs; β-actin was a loading control. (j) Rab6a and NMIIA-P-S1943 Western blot of the complexes pulled down with NMIIA-P-S1943 Ab VA-13 cells treated with scramble or GOLGB1 (giantin) siRNAs; input was normalized by Rab6a.
