Figure 5.

JNK and basal autophagy. A375 cells were exposed to SP600125, and JNK activation (P-JNK) was evaluated by western blotting analysis (a; JNK was used as the loading control). A375 cells expressing a p62-GFP recombinant protein were treated or untreated with bafilomycin A (Baf, 4 h) and SP600125 (6 h) alone or in combination, and the occurrence of autophagy was analysed by measuring p62-GFP levels by citofluorimetric analysis (b; n=3), LC3 conversion by western blotting analysis (c; Gapdh was used as the loading control), and by evaluating the presence of p62-GFP cytosolic puncta by confocal analysis (d; bar=10 μm). A Flag-tagged JNK dominant negative (JNK-DN) was ectopically expressed in A375 cells by transient transfection and expression levels of JNK-DN protein and LC3 conversion, and accumulation was evaluated in presence or absence of Baf by western blotting (e). GFP or BRAFV^600E expressing SK-Mel-110 cells were transiently transfected with expression plasmids encoding Flag-tagged Beclin 1 and protein extracts were subjected to IP using an anti-Flag antibody. Purified complexes were analysed together with the corresponding total extracts by western blotting using anti-Flag (f, top), anti-Bcl-X_L (f, middle) and anti-Mcl-1 (bottom) antibodies. A375 cells were transiently transfected with expression plasmids encoding wild-type or a T69A/S70A/S87A mutant Bcl-2. Cells were treated or untreated with bafilomycin, as indicated, and total Bcl-2 protein expression together with LC3 conversion were evaluated by western blotting analysis (g; Gapdh was used as the loading control)