Compatible fluorescent labeled secondary antibodies

Cells or tissues were lysed on ice with ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc SC, and 2 mg/ml protease inhibitor mixture (Roche Diagnostics) and samples prepared. Total protein (25 μg) was loaded into SDS-PAGE followed by transfer to nitrocellulose membranes. Immunoblotting was performed at 4 °C overnight followed by 1 hr incubation with LI-COR compatible fluorescent-labeled secondary antibodies (LI-COR Biosciences). Bands were visualized on the Odyssey CLx platform (LICOR Biosciences). Quantifications were based on densitometry using ImageJ.

Cells or tissues were lysed on ice with ice-cold lysis buffer containing 50 mm Tris-HCl, pH 7.4, 0.1 mm EDTA, 0.1 mm EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mm NaCl, 10 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, 1 mm Pefabloc SC, and 2 mg/ml protease inhibitor mixture (Roche Diagnostics) and samples prepared. Total protein (25 µg) was loaded into SDS-PAGE followed by transfer to nitrocellulose membranes. Immunoblotting was performed with the following primary antibodies: LDLR (ab30532, Abcam), SR-B1(ab137829, Abcam), ALK-1 (70R-49334, Fitzgerald), DNM2 (ab65556, Abcam), GAPDH (2118, Cell Signaling Technology), eNOS (sc-7271, Santa Cruz Biotechnology), BMPR2 (612292, BD Biosciences), CAV-1 (610060, BD Biosciences), β-actin (sc-69879, Santa Cruz Biotechnology), HSP90 (610419, BD Biosciences). LI-COR compatible fluorescent-labeled secondary antibodies (LI-COR Biosciences). Bands were visualized on the Odyssey CLx platform (LI-COR Biosciences). Quantifications were based on densitometry using ImageJ.

Cells or tissues were lysed on ice with ice-cold lysis buffer containing 50 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc SC, and 2 mg/ml protease inhibitor mixture (Roche Diagnostics) and samples prepared. Total protein (25 μg) was loaded into SDS-PAGE followed by transfer to nitrocellulose membranes. Immunoblotting was performed at 4°C overnight followed by 1 h incubation with LI-COR compatible fluorescent-labeled secondary antibodies (LI-COR Biosciences). Bands were visualized on the Odyssey CLx platform (LICOR Biosciences). Quantifications were based on densitometry using ImageJ.