The Bax N-terminus or TM1 domain is not essential for association with Bcl-xL. A, HEK cells transfected with Bax-GFP, Bax 30–192GFP, Bax 30–146 GFP, Bax 30–105 GFP or RFP-Bcl-xL were harvested after 15 hours and imaged using confocal microscopy. Representative images (processed as described in methods) are shown. Scale bar: 10 microns B, HEK cells transfected with each of the constructs in A and RFP-Bcl-xL were harvested 15 hours post-transfection. Confocal images for distribution of Bax constructs (green) and RFP-Bcl-xL (red) were assessed for co-localization (Merge, right panel). Scale Bar: 10 microns. Representative images (each corresponding to a single image field) are shown. C, From the experiment in B, cells positive for GFP and RFP were scored for punctate or diffuse patterns of GFP by microscopy. The data are representative of two independent analyses. D, HEK cells transfected with Bax-GFP + Bcl-xL (left panel) or Bax 30–146-GFP + Bcl-xL (middle panel) or Bax 30–105-GFP + Bcl-xL (right panel) were harvested after 15 hours of culture. Bcl-xL was immunoprecipitated from cell lysates and the immunoprecipitates assessed by western blot analysis for Bax or the mutant constructs using an antibody to GFP and for the presence of actin. WCL represents the CHAPS buffer solubilized whole cell lysate, which was used for immunoprecipitation.
Parikh et al. BMC Cell Biology 2007 8:16 doi:10.1186/1471-2121-8-16