Secondary anti mouse igg hrp

Following passage, cells were first washed with PBS before being lysed with fresh RIPA buffer and centrifuged at 10,000× g for 10 min at 4 °C. Total protein concentration in the supernatant was measured using the BCA protein kit (Sigma-Aldrich). Western Blot analysis was performed on 30 μg of protein using DNMT3B (R&D System/MAB7646, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK, TET1 (ThermoFisher/GT1462, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK, HIF1A (Novusbio/NB100-479, Secondary Anti-rabbit IgG-HRP Cell Signalling/70745, London, UK, HIF2A (Novusbio/NB100-122, Secondary Anti-rabbit IgG-HRP Cell Signalling/70745), and GAPDH (Merck/MAB374, Secondary Anti-mouse IgG-HRP, Cell Signalling/70765, London, UK). The immunoreactivity bands were subjected to UpLight HRP chemiluminescent substrate solution (Uptima) and imaged using a FluorChem M Imager.

Cells (5 × 10⁵) were seeded in 60 mm plates, washed with PBS, trypsinized and transferred to a microcentrifuge tube. After centrifuging and washing in PBS, cells were lysed in RIPA buffer (Sigma, St. Louis, MO) containing phosphatase and protease inhibitor cocktail and 1 mmol/L phenylmethanesulfonyl fluoride (Thermo Scientific, Waltham, MA). Protein concentrations were measured using the BSA protein assay (Pierce, Thermo Scientific, Waltham, MA) per protocol. Equal amounts of protein were separated with 10% SDS-PAGE gel electrophoresis and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Chicago, IL). After blocking for 1 hour in Tris-buffered saline containing 0.1% Tween 20 and 5% milk, the membranes were probed with various primary antibodies: anti-TAGLN2 polyclonal antibody (Abcam, Cambridge, United Kingdom), anti-IDH1 R132H mutant antibody (Cell Signaling Technologies, Danvers, MA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (American Research Products, Waltham, MA). Membrane was then probed with secondary anti-rabbit IgG conjugated to horseradish peroxidase (HRP) and secondary anti-mouse IgG-HRP (Cell Signaling Technologies, Danvers, MA). Immunoreactivity was detected by enhanced chemiluminescent (ECL) kit (Amersham Biosciences Co., Little Chalfont, United Kingdom).