Journal: PLoS ONE

Article Title: Lysosomal Interaction of Akt with Phafin2: A Critical Step in the Induction of Autophagy

doi: 10.1371/journal.pone.0079795

Figure Lengend Snippet: A . PIP strips were incubated with GST-Phafin2 WT, PH, or FYVE and the binding ability was examined by GST immunoblot. A comparable amount of recombinant wild type, N-term (PH domain), or C-term (FYVE domain) of Phafin2 was used (left panel). B . PtdIns(3)P interacting defective-Phafin2 (R53C, R171A, and R172A) failed to bind to PtdIns(3)P on PIP strip (right panel). C . BiFC analysis, 3-MA or wortmannin treatment abrogated the accumulation of the Akt-Phafin2 protein complexes at perinuclear lysosome after rapamycin treatment. D . Mutant Phafin2 displayed no accumulation of perinuclear lysosome after rapamycin treatment (right panels). E – F . Mutant Phafin2 failed to induce autophagy determined by decreased intensity of LC3-II band ( E , lane 6). The percentage of autophagy inhibition was 29.3±7.50% out of three independent experiments. The observation was consistent, as determined by the absence of GFP-LC3 puncta using confocal microscopy ( F , lower right side panels). Note that ectopic expression of wild type Phafin2 modestly enhanced autophagy induction determined by LC3-II blot (E, lane4 upper panel) and LC3 puncta (F. lower panels). White scale bar represents 10 µm.

Article Snippet: Twenty five nanograms of the indicated recombinant protein in 3% blocking buffer were incubated with PIP-STRIPS (Echelon Bioscience, P-6001) for 2 hr at room temperature and subsequently immunoblotted using anti-GST antibody (GE healthcare, #27-4577-01).

Techniques: Incubation, Binding Assay, Western Blot, Recombinant, Stripping Membranes, Mutagenesis, Inhibition, Confocal Microscopy, Expressing