Skip to main content
. Author manuscript; available in PMC: 2014 Apr 11.
Published in final edited form as: Mol Cell. 2013 Feb 21;50(1):29–42. doi: 10.1016/j.molcel.2013.01.022

Figure 4. Association of p110β with Rab5 is required for Rab5 activity and autophagy.

Figure 4

(A) HEK293T cells were transfected with Myc-tagged p110α or p110β constructs together with Flag-p85α. 48 h later, cell lysates were subjected to pull-down with GST or GST-Rab5 loaded with GTPγS. (B) p110β-Q596C and p110β-I597S possess intact kinase activity. The lipid kinase activity assay was performed as previously described (Dbouk et al., 2010). Data presented are the means of three independent experiments, normalized against that of p110β protein levels. Error bars: SEM. (C) HEK293T cells were transfected with indicated constructs. 48 h later, cell lysates were subjected to immunoprecipitation with Myc-antibody conjugated to agarose. Blots of the precipitates and the input were probed with Myc and Flag antibodies. (D) β−/− MEFs were stably reconstituted with indicated constructs and were analyzed for indicated proteins. (E) Lysates of stably reconstituted MEFs were subjected to GST-R5BD pull-down assays to determine the amount of Rab5-GTP. The relative amount of Rab5-GTP against total Rab5 is shown. Error bars: SEM; n=3; * p<0.05. (F) MEFs were analyzed for p62. The relative amount of p62 is shown. Error bars: SEM; n=3; * p<0.05. (G) MEFs as indicated were incubated with [14C]valine for 24 h. The cells were left untreated or serum-starved for 4 h. Degradation of long-lived proteins was measured. Error bars: SEM; n=3; * p<0.05. (H) MEFs generated as in (D) were cultured in complete or serum-free medium for 6 h and treated with or without bafilomycin A1 (50 nM). Western blots of cell lysates were probed for LC3 and β-tubulin. Quantification of LC3-II against β-tubulin is shown. Error bars: SEM; n=3; * p<0.05. (I) MEFs generated as in (D) were transfected with GFP-LC3. 48 h later, cells were cultured in complete or serum-free medium for 4 h, in the absence or presence of bafilomycin A1 (50 nM). Representative images of cells without bafilomycin A1 treatment are shown. Scale bar: 20 μm. Quantification of autophagic cells is shown on the right. Data are average values of at least 4 blind countings with over 200 cells. Error bars: SD; * p<0.05; ** p<0.01.