Anti lamin b antibody

Nuclear proteins from MA-10 Leydig cells were extracted as previously described [47 (link)] and quantified using a Bradford protein assay (Bio-Rad Laboratories, Mississauga, ON, Canada). In vitro translated COUP-TFII was generated using the Promega TnT quick coupled transcription/translation system (Thermo Fisher Scientific) according to the manufacturer’s protocol without additional purification or quantification. Denatured proteins were resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Etobicoke, ON, Canada). Immunodetection was performed using horseradish peroxidase–conjugated antibodies according to the manufacturers’ protocols and the following reagents: ECL and ECL Prime Western blotting detection reagents (GE Healthcare Life Sciences, Mississauga, ON, Canada), Clarity Western ECL substrate (Bio-Rad Laboratories), Clarity Max Western ECL substrate (Bio-Rad Laboratories). Detection of COUP-TFII and lamin B proteins was performed using a mouse monoclonal anti–COUP-TFII antibody (dilution 1:1000; R&D Systems, Minneapolis, MN; catalog no. PP-H7147-00, RRID:AB_2155627) [48 ] and a goat polyclonal anti–lamin B antibody (dilution 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA; catalog no. sc-6216, RRID:AB_648156) [49 ], respectively.

Sample cells, which were treated with or without DFO (100 μM) for 6 h, were harvested and suspended in low salt buffer [10 mM HEPES, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, protease inhibitor cocktail (PIC; Roche Diagnostics), and 1% Nonidet P-40] and nuclear pellets were collected by centrifugation. The nuclear pellets were suspended in high salt buffer (20 mM HEPES, 400 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA, PIC); after centrifugation, a nuclear extract was obtained. These nuclear extract samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel by electrophoresis and transferred onto PVDF membrane (Millipore). An immunoblotting analysis was performed as previously described [12 (link)]. Anti-human HIF-2α antibody [14 (link)], anti-human HIF-1α antibody (Novus Biologicals), and anti-Lamin B antibody (Santa Cruz Biotechnology, Inc.) were used for the immunoblotting assay. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) or goat anti-mouse IgG (Life Technologies) was used as the secondary antibody, and enhanced chemiluminescence (GE Healthcare Biosciences) was used for detection.