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. Author manuscript; available in PMC: 2019 Nov 20.
Published in final edited form as: Circulation. 2018 Nov 20;138(21):2395–2412. doi: 10.1161/CIRCULATIONAHA.118.034083

Figure 3. Lack of a role of p115 RhoGEF, Rac1, RhoA and Pak2 in thrombin- induced CD36 expression and foam cell formation.

Figure 3.

A. Equal amounts of protein from control and the indicated time periods of thrombin-treated cells were immunoprecipitated with anti-p115 RhoGEF or anti-Pak2 antibodies and immunocomplexes were analyzed by Western blotting using anti-PY20 or anti-pSer/Thr antibodies, respectively. For GTP-Rac1 or GTP-RhoA levels, equal amounts of protein from control and the indicated time periods of thrombin- treated cells were incubated with GST-Pak1 or GST-Rhotekin-conjugated glutathione sepharose 4B beads and the pull down proteins were analyzed by Western blotting for Rac1 and RhoA levels, respectively. The same cell extracts were also analyzed by Western blotting for total Rac1 and RhoA levels. B & C. Cells were transfected with siControl or sip115 RhoGEF(100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 expression and foam cell formation, respectively. The CD36 blot was reprobed for p115 RhoGEF or β-tubulin levels to show the effect of the siRNA on its target and off target molecules levels. D & E. Cells were transfected with siControl or siRac1 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for Rac1 or β-tubulin to show the effect of the siRNA on its target and off target molecules levels. F & G. Cells were transfected with siControl or siRhoA (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for RhoA or β-tubulin to show the effect of the siRNA on its target and off target molecules levels. H & I. Cells were transfected with siControl or siPak2 (100 nM), quiesced, treated with and without thrombin for 1 hr or 4 hrs and analyzed for CD36 levels and foam cell formation, respectively. The CD36 blot was reprobed for Pak2 or β-tubulin to show the effect of the siRNA on its target and off target molecules levels. The bar graphs represent Mean ± S.D. values of three experiments. *, p < 0.05 vs control or siControl. Scale bar is 50 μm.