Figure 5
EtOH effect on Rab6a: the loss of dimer form, reduction of interaction with giantin and enhancement of association with NMIIA. (a,b) Proximity ligation assay (PLA) of Giantin/Rab6a and Rab6a/NMIIA-P-S1943 in control VA-13 cells and treated with 35 mM EtOH for 72 h (a) and in hepatocytes from control and EtOH-fed rats (b) as analyzed by confocal microscopy; bars, 10 μm. (c) Quantification of PLA red spots (close proximity sites) scored per cell with more than one spots; n = 60 cells. The results are presented as a mean ± SD from three independent experiments; *p < 0.001. (d) Rab6a Western blot of the human active Rab6a recombinant protein and its IP with anti-Rab6a Ab. One μg of Rab6a protein was used as an input. (e) Rab6a Western blot of the cell lysate of VA-13 cells transiently transfected with empty PCMV vector (Ctrl) or with Rab6(T27N) plasmid; β-actin was a loading control. (f) Schema of the in vitro Rab6a capturing experiment. Anti-giantin-Ab was exposed to Dynabeads M-270 (step 1) followed by incubation with cell lysate of VA-13 cells overexpressing Rab6aT27N (step 2) and active Rab6a protein (step 3). The beads containing the complex anti-Giantin Ab-Giantin-Rab6a were: served as a control (fraction a), incubated with cell lysate of VA-13 cells overexpressing Rab6aT27N and treated with NMIIA siRNAs (fraction b) and incubated with cell lysate of VA-13 cells overexpressing Rab6aT27N (fraction c). (g) Giantin and Rab6a Western blot of the fractions a, b and c, as described in f. Top panel indicates the input for fractions b and c. (h) Rab6a Western blot of the Golgi fraction from control and EtOH-treated VA-13 cells and hepatocytes from control and EtOH-fed rats. The results shown are representative of three independent experiments.
