Figure 4.

Dab2 inhibits chronic TGF-β-induced autophagy by blocking Vps34–Beclin-1 interaction and promotes apoptosis by attenuating ERK–Bim interaction. (a) Beclin-1 immunoprecipitation using whole-cell lysates from cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Dab2, Vps34, Bim, ERK and p-ERK. (b) Immunofluorescence analysis of Dab2 and Beclin-1 in NMuMG cells treated with TGF-β for the times indicated. Photos were taken by confocal microscope. Scale bars, 10 µm. (c) Co-immunoprecipitation analysis using anti-ERK1/2 antibody from whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of p-ERK1/2 and Bim. (d) Immunofluorescence analysis was performed to detect the co-localization between ERK and Bim before and after NMuMG cells were treated for 7 days. Scale bars, 10 µm. (e) Top: p-ERK1/2 immunoprecipitation using whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Bim. As a control, p-Bim ubiquitylation and degradation is inhibited by PYR-41, a cell-permeable inhibitor of the ubiquitin-activating enzyme E1 (20 µM, 4 h before cell lysates were collected). Bottom: immunoblotting analysis was employed to detect the Bim and p-Bim expression from whole-cell lysates of NMuMG cells treated with TGF-β for the times indicated in the presence of 20 µM PYR-41. (f) Immunofluorescence analysis was performed to detect the co-localization between p-ERK and Bim before and after NMuMG cells were treated for 7 days in the presence of PYR-41. Scale bars, 10 µm. The experiments were repeated three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.