Irdye 800 conjugated secondary antibody

To determine HA-DMT1 phosphorylation, cells transfected with HA-DMT1 or empty vector were scraped into ice-cold PBS and pelleted by centrifugation, then lysed in Nonidet-P40 (NP-40) buffer (40 mM HEPES, pH 7.4, 120 mM NaCl, 5% glycerol, 1% NP-40 and 1 mM EDTA, pH 8.0) containing phosphatase inhibitors (10 mM pyrophosphate, 10 mM β-glycerophosphate, 50 mM NaF and 0.5 mM orthovanadate) for 30 min at 4°C with rotation. Samples were centrifuged for 10 min at 15 000×g, and the protein concentration of the lysates was determined by the Bradford assay. The cell lysates were preincubated with anti-HA agarose (Pierce) overnight at 4°C with rotation and washed with TBS before protein elution at 95°C for 5 min. Eluates were electrophoresed on 4–20% gradient gels, transferred to PVDF membranes and the extent of HA-DMT1 phosphorylation was assessed by immunoblotting with mouse antiphosphoserine antibody (Millipore) followed by IRDye 800-conjugated secondary antibodies (Rockland Immunochemicals). Meanwhile, total cell lysates were immunoblotted with either anti-HA antibody or by antiactin antibody followed by IRDye800-conjugated secondary antibodies. After further washing, immunoreactivity was detected by an Odyssey Infrared Imaging System (Li-COR).

Cells were washed twice with ice-cold PBS, and the total cell lysates were prepared in lysis buffer containing 150 mM NaCl, 1 mM EDTA, 50 mM Tris and 1% Triton containing a protease inhibitor cocktail (Complete Mini, Roche). Protein concentration was determined by the Bradford assay. Samples (25 µg) were separated by electrophoresis on 10% SDS–PAGE gels and transferred to PVDF membranes, and immunoblotted with rabbit anti-CB1 or -CB2 antibody (Cayman Chemical Company). The primary antibody was detected with IRDye 800-conjugated secondary antibodies (Rockland Immunochemicals, Inc.). After further washing, immunoreactivity was detected by an Odyssey Infrared Imaging System (Li-COR).