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. 2008 Oct;19(10):4188–4200. doi: 10.1091/mbc.E07-10-1089

Figure 7.

Figure 7.

Expression of dominant-negative Rab11a, Gαi2, or PTX treatment prevents the antiapoptotic and proliferative effect of LPA. (A) Antiapoptotic effect of 10 μM LPA was assessed by Annexin V binding in HEK-293T cells transfected with wild-type, constitutively active, or dominant-negative GFP-tagged mutants of Rab11a (Rab11a WT, Rab11a Q70L, or GFP-Rab11a S25N, respectively), Gαi2, or PTX treatment. NS, nonstimulated cells. The fraction of apoptotic cells in vector-transfected nonstimulated cells was normalized to 100%. Error bars represent the means ± SEM of three independent experiments (analysis of variance and Student-Newman-Keuls test). *p < 0.001 and **p < 0.05 compared with nonstimulated cells. (B) Proliferative effect of 10 μM LPA was assessed by BrdU labeling in HEK-293T cells transfected with wild-type, constitutively active, or dominant-negative GFP-tagged mutants of Rab11a (WT, Q70L, and S25N, respectively), Gαi2, or PTX treatment. NS, nonstimulated cells. Error bars represent the means ± SEM of four independent experiments done by triplicate (analysis of variance and Student-Newman-Keuls test). *p < 0.01 and **p < 0.001 compared with nonstimulated cells. (C) Effect of LPA and PTX on the recruitment to endosomes and phosphorylation of GSK3-β. Total cell lysates and endosomal fractions from samples shown in the Figure 6C were used to detect the presence and endosomal phosphorylation of GSK3-β by using specific antibodies. NS, nonstimulated cells. (D) Rab11 knockdown interferes with the antiapoptotic effect of LPA. Antiapoptotic effect of 10 μM LPA was assessed by Annexin V binding in HEK-293T cells transfected with Rab11 shRNA. NS, nonstimulated cells. Error bars represent the means ± SEM of four independent experiments (analysis of variance and Student-Newman-Keuls test). *p < 0.05 compared with vector-transfected cells. (E) Rab11 knockdown does not globally disrupt LPA-dependent signaling pathways. HEK-293T cells transfected with Rab11 shRNA were serum starved and stimulated with 10 μM LPA at indicated times. Phosphorylation of AKT and ERK in total cell lysates was detected by Western blotting using phospho-specific antibodies. The expression of AKT, ERK, Rab11, and Rab5 was also detected by Western blot as indicated. A representative blot is presented.