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. Author manuscript; available in PMC: 2014 Mar 15.
Published in final edited form as: Cancer Res. 2013 Jan 14;73(6):1946–1957. doi: 10.1158/0008-5472.CAN-12-3710

Fig. 6. PP242 (A–C) or rictor knockdown (D–F) destabilizes FLIPS protein, whereas enforced expression of rictor stabilzes FLIPS (G and H).

Fig. 6

A, PP242 reduces FLIPS stability. H157 cells were treated with DMSO or 1 μM PP242 for 16 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/ml cycloheximide (CHX). At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantified with NIH Image J software (Bethesda, MA) and were normalized to actin. The results were plotted as the relative FLIPS levels compared to those at the time 0 of CHX treatment (bottom panel). B, The proteasome inhibitor MG132 inhibits FLIPS reduction by PP242. H157 and H1299 cells were pre-treated with 20 μM MG132 for 30 minutes prior to the addition of 1 μM PP242. After co-treatment for an additional 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, PP242 increases FLIPS ubiquitination. H1299 cells were co-transfected with HA-ubiquitin and Flag-FLIPS plasmids using Lipofectamine 2000 reagent for 24 h. The cells were then pre-treated with 20 μM MG132 for 30 minutes and then co-treated with 1 μM PP242 for another 4 h. Whole-cell protein lysates were then prepared for IP using anti-Flag antibody followed by Western blotting (WB) using anti-HA antibody for detection of ubiquitinated FLIPS (Ub-FLIPS) and anti-Flag antibody for detection of ectopic FLIPS. D and E, Rictor knockdown decreases FLIPS stability. H226 cells were transfected with control (Ctrl) or rictor siRNA for 48 h. Similar to the assay in panel A, the cells were treated with CHX and harvested at the given time post CHX treatment for preparation of whole-cell protein lysates and subsequent Western blotting to detect FLIPS. Rictor knockdown efficiency was demonstrated with Western blotting in D. F, Rictor knockdown increases FLIPS ubiquitination. H157-scramble and H157-shRictor cells were co-transfected with HA-ubiquitin and Flag-FLIPS plasmids using Lipofectamine 2000 reagent for 48 h followed with exposure to 20 μM MG132 for additional 4 h. Ubiquitinated FLIPS (Ub-FLIPS) was then detected as described in C. G and H, HEK293 cells were co-transfected with FLIPS and rictor expression plasmids for 48 h. The cells were harvested for Western blotting to detect the indicated proteins (G) or exposed to CHX as we did in panel A for FLIPS stability assay (H).