Bak ab 1

Artesunate, MG132, EB, and AO were purchased from Sigma-Aldrich Inc. Venetoclax (ABT-199) was purchased from Selleck Chemical. Cytarabine and Q-VD-OPh were purchased from MedChemExpress. Antibodies to PARP and caspase-8 were obtained from BD Biosciences. Antibodies to Bcl-2 (C-2), Actin (C-2), Mcl-1 (S-19), Mcl-1 (G-7), Bax (6A7) and Chk1 (G-4) were obtained from Santa Cruz Biotechnology, Inc. Antibodies to Mcl-1 (D35A5), Bim (C34C5), Bak (D4E4), Bax poly, Noxa (D8L7U), Cleaved Caspase-3 (Asp175), Phospho-Chk1 (Ser345) (133D3), and Phospho-Histone H2A.X (Ser139) (20E3) were obtained from Cell Signaling Technology, Inc. Antibodies to Noxa were obtained from Abcam, Inc. Bak (Ab-1) was obtained from Merck Millipore. NOXA(sc-37305), BIM(sc-29802), MCL1(sc-35877)siRNA, and a control siRNA were purchased from Santa Cruz Biotechnology, Inc. NOXA(s10708), BIM(s195011), MCL1(s8583)siRNA was purchased from Thermo Fisher Scientific.

Peptides for BIM (MRPEIWIAQELRRIGDEFNA), BID (EDIIRNIARHLAQVGDSMDRY), NOXA (AELPPEFAAQ LRKIGDKVYC), PUMA (EQWAREIGAQLRRMADDLNA), HRK (WSSAAQLTAARLKALGDE LHQ), MS-1 (RPEIWMTQGLRRLGDEINAYYAR), BAD (LWAAQRYGRELRRMSDEFEGSFKGL) and PUMA-2 A (EQWAREIGAQARRMAADLNA) were from New England Peptide (Gardner, MA, USA) or GenScript (Piscataway, NJ, USA). Antibodies against MCL-1, BAK and GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA, USA); OPA1, DRP-1, HSP60 and Cytochrome C from BD Biosciences (Oxford, UK); Tubulin, MFN1, MFN2, Caspase-3, MCL-1 (RC-13) from Abcam (Cambridge, UK); BAK (Ab-1) from Merck Chemicals Ltd (Nottingham, UK) were used. For bacterial expression of recombinant proteins, a human MCL-1 dsDNA corresponding to amino acids E173- R329 was amplified using the primer sets, 5′-AAGTTCTGTTTCAGGGCCCGGAGTTGTACCGGCAGTCG-3′ and 5′-ATGGTCTAGAAAGCTTTACCTGATGCCACCTTCTAGGTC-3′ and cloned into pOPIN F (OPPF-UK, Oxford, UK) to generate N-terminally His6-tag fusion protein. This was used as a template to generate the R263A mutant using site-directed mutagenesis with the primer sets, 5′-CGTAACAAACTGGGGCGCGATTGTGACTCTC-3′ and 5′-GAGAGTCACAATCGCGCCCCAGTTTGTTACG-3′. GFP-DRP-1 K38A plasmid was described previously.^{33 (link)} All other reagents, unless mentioned otherwise, were from Sigma-Aldrich Co. (St. Louis, MO, USA).