Fig. 4. BNIPXL-RhoA interaction requires an intact BCH domain and reduces active RhoA levels and serum-induced stress fiber assembly. (Ai) Schematic diagram of BNIPXL internal deletions: 0 (600-616), 1 (617-655) and 2 (656-684). All numbers are inclusive. Lysates expressing Flag-BNIPXL full-length, WBCH, 0, 1 and 2 with (Aii) HA-RhoA or (B) HA-BNIPXL were immunoprecipitated and probed with anti-HA (first panel) and reprobed with anti-Flag (second panel) to show the amounts of precipitated proteins. Protein expression was verified with anti-HA (third panel) and anti-Flag (fourth panel). The asterisk indicates the position of the expected bands. Arrow indicates the position of the light chain from antibody. (C) Lysates expressing (i) wild-type RhoA, Q63L, F30L or T19N mutants were incubated with GST or GST-RBD to confirm the specificity of the GST-RBD. Lysates expressing Flag-BNIPXL full-length or deletions with (ii) wild-type RhoA, (iii) Q63L or (iv) F30L mutants were incubated with GST-RBD. Bound proteins and WCL were analyzed using anti-RhoA (first and third panels, respectively) or anti-Flag for BNIPXL (fourth panels) before Amido Black staining to ascertain equal loading (second panels). (Di) 293T cells cotransfected with HA-BNIPXL and the shRNA constructs or scrambled negative control was lysed 72 hours post-transfection and subjected to anti-HA (100 µg WCL) (first panel) or anti-RhoA (50 µg WCL) (third panel) westerns to determine the extent of expression knockdown and endogenous RhoA levels, respectively and normalized against anti-tubulin (second and fourth panels). (Dii) 293T cells coexpressing wild-type RhoA, Q63L or T19N mutants were incubated with GST or GST-RBD to confirm the specificity of GST-RBD. Lysates expressing Flag-RhoA wild type with Flag-BNIPXL or the shRNA constructs were incubated with GST-RBD. Bound proteins and WCL were analyzed using anti-RhoA (first panel) or anti-Flag (third panel) to determine protein expression. Amido Black staining was used to ascertain equal loading of GST fusion proteins (second panel). Asterisks indicate position of expected bands. (Ei) Representative fields of NIH3T3 expressing full-length BNIPXL and BNIPXL deletions were starved 24 hours post transfection for 16 hours and stimulated with 0.5% serum-containing growth medium for 10 minutes. Cells were fixed, stained and visualized by immunofluorescence. Scale bars: 20 µm. (Eii) For quantitative analysis, the ratio of transfected cells with and without stress fibers was scored and at least 100 transfected cells were counted per sample per experiment. Data are means ± s.d. (n=3). Differences between values not sharing the same letters (a, b, c) are statistically significant at P<0.01 by analysis of variance and the Student-Newman-Keuls multiple range test (Statgraphics).