Anti sqrdl antibody

Cell lysates (20 µg) were separated on 12% polyacrylamide gels with semidry transfer to PVDF membranes (Millipore, Billerica, MA). Membranes were blocked and and incubated overnight with either anti-CSE (Gamma Cystathionase Antibody, Proteintech # 12217-1-AP, dil 1:500) or anti-SQR (Anti-SQRDL antibody, Abcam # ab71978, dil 1:500). Bands were visualised using chemiluminiscense detection (GE Healthcare Life Sciences). Following imaging, the anti-CSE and anti-SQR antibodies were stripped using stripping buffer and probed overnight with either β-actin (Sigma Aldrich # A5441, dil 1:2,500) or VDAC (Anti-VDAC1/Porin antibody, Abcam # ab34726, dil 1:2,500). β-actin and VDAC were used as a loading controls. Images were cropped using Image J software. Full-length membranes are included in the supplementary section.

The MC3T3-E1 cells and primary monocytes were lysed in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM of Tris buffer, pH 8.0). The lysates were centrifuged at 16,000×g for 20 min at 4° C to remove cellular debris. The protein concentration was determined using the Dc Protein assay kit (BIO-RAD; Hercules, CA, USA). Protein samples were heated at 95° C for 5 min and analyzed by SDS-PAGE on 12% polyacrylamide gels. The proteins were electroblotted onto PVDF membrane (Millipore; Concord Road Billerica, MA, USA). The membrane blots were blocked with 5% (w/v) BSA, incubated with primary and secondary antibodies, and then visualized by the WEST-ZOL plus ECL Western blotting detection system (iNtRON Biotechnology; Daejeon, Korea). Anti-SQRDL antibody was purchased from Abcam (Cambridge, UK) and anti-β-actin, HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated rabbit anti-goat IgG antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).