Fig. 3. BNIPXL binds to specific RhoA conformers. (A) Flag-BNIPXL full-length and fragments were transcribed and translated in vitro and incubated with GST or GST-RhoA (free of guanine nucleotides). Bound proteins were subjected to anti-Flag western blotting for short (30 seconds) and long (1 hour) exposure times. Amido Black staining was used to ascertain equal loading of GST fusion proteins. Asterisks indicate positions of the expected bands. (B) HEK293T lysates expressing Flag-BNIPXL fragments were incubated with unloaded GST-RhoA, GST-RhoA preloaded with GDP or GTPS. Bound proteins and WCL input were analyzed with anti-Flag or anti-Myc before Amido Black staining. (C) Lysates expressing Flag-BNIPXL (i) full-length, (ii) CBCH or (iii) WBCH fragments with vector control, HA-RhoA wild type or mutants were subjected to immunoprecipitation and bound proteins detected with anti-RhoA (first panel). Blots were reprobed with anti-Flag to show the amounts of precipitated proteins (second panel). Expression of RhoA (third panel) and BNIPXL (fourth panel) was verified. Asterisks indicate position of the expected bands. Arrows indicate position of the antibody light chain.