Fig. 5: PLEKHN1 replaces Bid in Bax-Bid complexes to form a Bax–Bak complex. HaloTag constructs were transfected into HT-29GFP or PLEK-KO+EGFP-PLEK cells

a Molecular interactions between PLEKHN1 and Bax or Bid. HaloTag protein complexes were pulled down and analyzed using a GFP antibody. Only HaloTag-Bid could pull down PLEKHN1 (lane 8). b GST-Bax and MBP-Bid-HA complexes were bound to GST-sepharose 4B.. The first elutions were obtained by adding purified PLEKHN1 protein. MBP-Bid-HA (Bid) was eluted without Bax (lane 2). The second elutions were obtained by adding the reduced glutathione, and indicated that similar amounts of proteins were bound to the columns. c The cell fraction analysis of HT-29, PLEK-KO, and PLEK-KO + EGFP-PLEK cells. Solb represents soluble proteins. TG treatment increased cytoplasmic translocation of cytochrome-c (lanes 4, 5, and 6). d Mito fraction represents mitochondrial proteins. In HT-29 cells, oligomerization of Bax had already occurred without TG-treatment (lanes 7, 10). The addition of PLEKHN1 to PLEK-KO cells restored the oligomerized Bax (lanes 9, 12). e-g To analyze the proximity of Bax or Bid, the mitochondrial fraction was obtained from the BMH-crosslinked protein samples, and loaded to 5–20% gradient gel with non-crosslinked protein samples. The different sizes of bands were labeled at the right end of Fig. 5g. e Immunoblot with anti-Bax antibody. The band A (~ 120 kDa) were observed every cross-linked samples. f Immunoblot with anti-Bid antibody. The major bands were seen in range C and G. g Immunoblot with anti-Bak antibody. The bands labeled with asterisk may be the undisolved large mitochondrial membrane fraction. The band B (~ 100 kDa) was overlapped with the band in 5e