Ni nta matrix

HeLa and SCC131 cells were obtained from National Centre for Cell Sciences, Pune, India. FBM cell line was kindly gifted by Dr. Milind Vaidya (ACTREC, India). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin were purchased from Hi Media, India. E. coli M15 bacterial strain and Ni-NTA matrix were purchased from Qiagen. All the salts were purchased from Sigma-Aldrich. Fura 2-AM, and JC-1 were purchased from Molecular probes Inc., USA. Antibodies were procured from Cell Signaling Technologies (CST), USA. Inverse pericam construct was kindly gifted by Dr. Atsushi, Miyawaki Riken, Japan.

Standard expression of the crARC and crARC cysteine to alanine mutants was performed in freshly transformed E. coli TP1000. Cells were grown aerobically in LB medium to an OD₅₅₀ equal to 0.1 before induction. TP1000 cells were induced with 10 µM isopropyl-β-d-thiogalactopyranoside (IPTG) and additionally supplemented with 0.1 mM sodium molybdate to initiate recombinant expression. BL21 cells were induced with 100 µM IPTG for expression. crCyb5-1 and crCytb5-R were purified as described previously [17 (link)]. Cells expressing proteins with heme groups were supplemented with 1 mM aminolevulinic acid to support heme synthesis. After induction, the cells were grown for 36 h at 22 °C. Purification of recombinant proteins was performed by the Ni-nitrilotriacetic acid (Ni-NTA) matrix, as recommended by the supplier (Qiagen), under native conditions at 4 °C, using minimal volumes of washing buffers to reduce dissociation of bound Mo from the proteins. The protein fractions were analyzed by SDS-polyacrylamide gel electrophoresis and only the pure fractions were taken and immediately desalted on a PD10 gel filtration column previously equilibrated with 100 mM Tris-HCl, pH 7.2. Protein concentration was determined by UV absorption measurements using the calculated extinction coefficient [43 (link)] of the analyzed polypeptides.

His-tagged PARP12-Mutants were generated from Human PARP12 cDNA from Origene as the pCMV6-XL5 using the oligonucleotides reported in Supplementary Table 1. The resulting pET28a plasmids encoding for His-PARP12-MUT1, His-PARP12-MUT2 and His-PARP12-MUT3 were used to transform E. coli strain BL21(DE3). The bacteria were grown in LB medium until they reached an OD₆₀₀ nm of 0.6 and were induced by the addition of 0.5 mM IPTG for 16 h at 20 °C. The proteins were then purified on Ni-NTA matrix (Qiagen) according to the manufacturer’s instructions.