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Figure 2


Fig. 2. The BNIPXL BCH domain associates with RhoA and RhoC but not RhoB, and elicits cell morphological changes. (A) Schematic diagram of BNIPXL and its fragments. Two splicing isoforms of full-length, FL{alpha} and FLβ; N-terminus without BCH, WBCH; N-terminus without CBCH, NBCH; C-terminus, C{alpha} and Cβ and C-terminus containing BCH, CBCH{alpha} and CBCHβ. (B) Lysates expressing Flag-BNIPXL with HA-vector control, (i) HA-RhoA or (ii) HA-Cdc42 were immunoprecipitated (IP) with anti-Flag and bound proteins immunoblotted (IB) with anti-HA (first panel). Blots were reprobed with anti-Rho GTPase (third panel) and anti-Flag (second panel) to show the amounts of precipitated proteins. Protein expression was verified using anti-HA for GTPases (first panel) or anti-Flag for BNIPXL (second panel) in whole cell lysates (WCL). Asterisks indicate position of the expected bands. Arrows indicate the position of the light chain of the antibody. (C) Lysates expressing Flag-CBCH with HA-RhoA, RhoB or RhoC were subjected to immunoprecipitation and bound proteins detected with anti-HA (first panel). Blots were reprobed with anti-Flag to show the amounts of precipitated proteins (second panel). Expression of Rho (third panel) and BNIPXL (fourth panel) was verified. Asterisks indicate the position of the expected bands. Arrows indicate the antibody light chain. (D) Lysates expressing HA-BNIPXL fragments with Flag-BNIPXLβ were immunoprecipitated, and bound proteins detected with anti-HA (first and third panels) followed by anti-Flag (second and fourth panels) to show amounts of precipitated proteins. Protein expression was verified with anti-HA (first and third panels) and anti-Flag (second and fourth panels), respectively. (E) HeLa cells expressing Flag-BNIPXL fragments in normal growth medium were fixed 24 hours post transfection and stained as indicated. F-actin was detected with TRITC-conjugated phalloidin and cells were visualized by confocal fluorescence microscopy. Arrows indicate membrane protrusions and protein concentrated at the tips of protrusions. Scale bar: 50 µm.





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