Goat anti rabbit ir800

Tissue or cell lysates were prepared by homogenization in a buffer containing: 10 mM HEPES (pH 7.9), 15 mM MgCl, 10 mM KCl, 0.5 mM EDTA, 0.2% Triton X-100, 1 mM benzamidine, 200 mg/ml aprotinin, 200 mg/ml leupeptin, 1 mM PMSF (Sigma-Aldrich) and phosphatase inhibitors: 20 mM b-glycerophosphate, 10 mM NaF, 10 mM sodium pyrophosphate, and 2 mM orthovanadate (Sigma-Aldrich). Tissue lysates were resolved by 8% (m/v) SDSPAGE in reducing conditions and transferred to nitrocellulose membranes. After blocking with 2% BSA, membranes were washed and incubated with an anti-pSMAD3, αSMA or α/β-Tubulin antibodies (Cell Signaling) overnight at 4°C. Proteins were detected with secondary Abs goat anti-mouse A680 (Life Technologies) and goat anti-rabbit IR800 (Thermo scientific) and visualized with an infrared imaging system (Odyssey; LI-COR Biosciences).

Cells were lysed in 1% Triton X-100, 0.5% n-dodecyl-β-d-maltoside, 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1 mM NaF, and 1 mM sodium orthovanadate containing protease inhibitors. Samples were separated by NuPAGE 4–12% Bis-Tris gel electrophoresis (Life Technologies). For immunoprecipitation, lysates were immunoprecipitated with anti-Cav1 rabbit Ab (BD Biosciences) coupled to Dynabeads protein A (Life Technologies). Immunoprecipitates were washed with lysis buffer, and proteins were transferred onto polyvinylidene difluoride Immobilon-FL membranes (Millipore). Membranes were incubated in blocking reagent (LI-COR Biosciences) before Western blotting analysis with anti-Cav1 rabbit Ab (BD Biosciences) or anti-Cav2 mouse mAb (clone 65; BD Biosciences). Proteins were detected with secondary Abs goat anti-mouse A680 (Life Technologies) and goat anti-rabbit IR800 (Thermo scientific) and visualized with an infrared imaging system (Odyssey; LI-COR Biosciences). Fractionation using a subcellular fractionation kit was performed according to the manufacturer’s instructions (Thermo Scientific).