Skip to main content
. 2016 Jul 7;7(32):52239–52254. doi: 10.18632/oncotarget.10469

Figure 2. VPS34 activates mTORC1/S6K1, leading to the increase in cell size (referring to Figure 1C for the clones stably expressing vector, VPS34-WT and VPS34-H868R).

Figure 2

A. WCL obtained from indicated stable cell clones, vector, WT_118 and H868R_07 was subjected to western blotting to monitor the phosphorylated and total expression levels of S6K1. B. Stable Vector, WT_118 and H868R_07 cells were assessed for phosphorylation of 4EBP1 at T37/46 position by Western blot analysis. Insulin-treated (100 nM for 30 min) NIH3T3 cells were used as a positive control for 4EBP1 phosphorylation. The membrane was stripped and re-probed for total 4EBP1. C. Phosphorylation of Erk1/2 and Akt (S473) were detected in stable cell clones as indicated in 1% and 10% serum containing media. Membranes was stripped and re-blotted with total Erk1/2 and Akt proteins. D. The relative cell size of the indicated stable clones was analyzed using a BD FACSCalibur flow cytometer to determine the mean FSC-H of cells for the measurement of relative cell size. E. Quantitative data of flow cytometric cell size were performed from two or more independent experiments and presented in the form of bar graph (**, p < 0.01).