Journal: Cell death discovery

Article Title: The membrane insertion of the pro-apoptotic protein Bax is a Tom22-dependent multi-step process: a study in nanodiscs.

doi: 10.1038/s41420-024-02108-x

Figure Lengend Snippet: Fig. 2 The GALLL motif in Hα1 was responsible for nanodisc-induced Bax precipitation and interacted with Tom22. A BaxWT and the constitutively active mutant BaxR9E were synthesized in the absence of nanodiscs. Reaction mixes were centrifuged (20,000 × g, 15 min) and equal volumes of the supernatants were added or not with 3.5 µM nanodiscs. 1 µg anti-Bax 6A7 or 2D2 antibodies (Santa-Cruz Biotechnology) were added and samples were incubated overnight at 4 °C under gentle agitation. 50 µL Protein-G sepharose beads (Cytiva) were washed with buffer IP (20 mM Tris/HCl pH 8.0, 200 mM NaCl) and were added for 3 h. The suspensions were then washed 5 times with 200 µL of buffer WIP (25 mM Tris/HCl pH 7.4, 500 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol) and twice with the same buffer diluted 10 times. Bound proteins were recovered by adding 30 µL of Laemmli buffer (without β-mercaptoethanol) and heating at 65 °C for 15 min. Samples were analyzed by SDS-PAGE and western blot against Bax, with a distinct antibody from the ones used for immunoprecipitations (Abcam). The 2D2 antibody did not immunoprecipitate the Bax R9E mutant. The figure is representative of 3 independent experiments. B BaxWT and BaxΨ (deleted of residues 1–19) were synthesized in the absence or in the presence of nanodiscs and analyzed like in Fig. 1A. Western blots were done with the Abcam antibody of which the undisclosed epitope is away from the N-terminus. Blots are representative of 4 independent experiments. C Supernatants from BaxΨ in (B) were analyzed as in Fig. 1C, showing the absence of BaxΨ-association to nanodiscs. The blot is representative of 2 independent experiments. D BaxΨ was co-synthesized with Tom22 in the presence of nanodiscs, and samples were analyzed like in (B). Blots are representative of 5 independent experiments. E Supernatants from (D) were analyzed like in (C), showing the partial association of BaxΨ to nanodiscs. Blots are representative of 3 independent experiments. F Representation of the Hα1 of Bax in the 1F16 structure [3]. Hydrophilic residues are in blue and hydrophobic residues are in red. G Same experiment as in Fig. 1A with BaxWT, BaxA24R, and BaxL26Q, showing the absence of precipitation of BaxA24R and BaxL26Q. Blots are representative of 2 independent experiments. H Same experiment as in Fig. 1C with BaxA24R showing its absence of association to nanodiscs. Blots are representative of 2 independent experiments.

Article Snippet: 1 μg anti-Bax 6A7 or 2D2 antibodies (Santa-Cruz Biotechnology) were added and samples were incubated overnight at 4 °C under gentle agitation.

Techniques: Mutagenesis, Synthesized, Incubation, Gentle, SDS Page, Western Blot