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. Author manuscript; available in PMC: 2018 Nov 16.
Published in final edited form as: Biochem J. 2017 Nov 16;474(23):3903–3914. doi: 10.1042/BCJ20170279

Figure 6. Stimulation of PI3Kβ by CA-Rac1 and LPA requires macropinocytosis.

Figure 6

(A) Stable p110β knockdown cells were transiently transfected with wild-type p110β, p110βKK-DD or p110βRBD, without or with GFP-CA-Rac1. After 48 h, cells were treated with 25 μM EIPA for 3 h, lysed and blotted for p110β, Akt and pT308-Akt. (B) Quantitation of pAkt/Akt immunoblots in Figure 6A. The data represent the mean ± SEM from three independent experiments. pAkt/Akt ratios in each experiment were normalized to that seen in cells expressing wild-type p110β and transfected with GFP-CA-Rac-1, but treated with 0.1% DMSO. (C) Stable p110β knockdown cells were transiently transfected with wild-type p110β without or with GFP-CA-Rac1. After 48 h, cells were treated for 15 min with 0.5 μM Latrunculin B, lysed and blotted for p110β, Akt and pT308-Akt. (D) Quantitation of pAkt/Akt immunoblots in Figure 6C. pAkt/Akt ratios in each experiment were normalized to that seen in cells expressing wild-type p110β and GFP-CA-Rac1. The data represent the mean ± SD from two independent experiments. (E) Stable p110β knockdown cells were transiently transfect with wild-type p110β, p110βKK-DD or p110βRBD. After 48 h, cells were starved overnight, treated with 25 μM EIPA for 3 h, and stimulated for 5 min with 10 μM LPA. Cells were lysed and blotted for p110β, Akt and pT308-Akt. (F) Quantitation of pAkt/Akt immunoblots in Figure 6E. pAkt/Akt ratios in each experiment were normalized to that seen in cells expressing wild-type p110β and stimulated with LPA but treated with DMSO. The data represent the mean ± SEM from three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Note: The western blot data in Figures 4A and 6A are taken from the same set of experiments, as are the data in Figures 3C and 6E. In each case, cells were treated with 0.1% DMSO or 25 μM EIPA. The EIPA arms of these experiments are presented separately in Figure 6, for clarity of the narrative. Because of this, blots for the controls (cells expressing wild-type p110β and treated with DMSO) are shown in Figures 4 and 3, and are not reproduced in Figure 6. We have included the quantitation of the controls (labeled DMSO) from Figures 3D and 4B in the bar graphs shown in Figure 6B,F, to facilitate a comparison of DMSO- versus EIPA-treated cells.

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