Skip to main content
. 2016 May 31;36(12):1803–1817. doi: 10.1128/MCB.00067-16

FIG 5.

FIG 5

Regulation of B-Myb by VEGF and PDGF. (A) VEGFR and PDGFR inhibitor prevents tyrosine phosphorylation of B-Myb. 786-O cells stably expressing 3×HA–B-Myb or control cells were sparsely cultured for 1 day, followed by RTK inhibitor treatment for 1 day. 3×HA–B-Myb was immunoprecipitated (IP) and immunoblotted (IB) with anti-pY or anti-HA antibody. (B) The intensities of tyrosine-phosphorylated 3×HA–B-Myb bands in panel A were normalized to those of total immunoprecipitated 3×HA–B-Myb bands, and mock treatment was set as 1. Data represent means ± SD from three independent experiments. (C) VEGFR and PDGFR inhibitor destabilizes B-Myb. 786-O cells stably expressing 3×HA–B-Myb and 3×FLAG-pVHL were sparsely cultured for 1 day, followed by RTK inhibitor treatment for 1 day. Then, cells were exposed to CHX (50 μg/ml) for 15 or 30 min. The lysates were subjected to Western blotting with antibodies against HA or FLAG. CBB staining was used as a loading control. (D) The intensities of 3×HA–B-Myb bands in panel C were normalized to those of CBB-stained bands. The amount of 3×HA–B-Myb at the beginning of the chase was set as 1. Data represent means ± SD from three independent experiments. (E) B-Myb interacts with VEGFR1. 786-O cells stably expressing 3×HA–B-Myb or control cells were sparsely cultured for 2 days. 3×HA–B-Myb was immunoprecipitated and immunoblotted with antibodies against VEGFR1 or HA. (F) Downregulation of VEGFR1, PDGFRα, and EGFR by confluent culture conditions. pVHL-reintroduced 786-O cells were cultured under confluent or sparse conditions overnight with or without MG132 (10 μM) or bafilomycin A1 (0.5 μM). The lysates were subjected to Western blotting with antibodies against VEGFR1, PDGFRα, or EGFR. Ponceau S staining was used as a loading control. (G) The intensities of VEGFR1, PDGFRα, or EGFR bands in panel F were normalized to those of Ponceau S-stained bands. The amount of VEGFR1, PDGFRα, or EGFR expressed under sparsely cultured conditions was set as 1. Data represent means ± SD from three independent experiments.