A00682

Tissue samples were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA pH 8.0, 1% NP-40) containing 1 mM phenylmethanesulfonyl fluoride (PMSF). The mixture was ground into homogenate on ice, and the supernatant was collected by centrifuging at 16,200 × g for 15 minutes at 4 °C. The protein concentration in samples was determined using a bicinchoninic acid assay kit (P0012S, Beyotime). Lysates were mixed with loading buffer and boiled for 10 minutes. An equal amount of proteins (30 µg) were run on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore MA). The membrane was blocked with 5% nonfat milk in TBST buffer (50 mM Tris, 1.37 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, pH 8.0) for 1 hour at room temperature. The membranes were then incubated with anti-tdTomato primary antibody (1:500; A00682, GenScript) or anti-GAPDH primary antibody (1:1000; AF1186, Beyotime) overnight at 4 °C. After washing three times with TBST buffer, the membrane was incubated with a secondary antibody (1:5000; Alexa Fluor790 Goat Anti-Rabbit, Jackson ImmunoResearch) for 1 hour at room temperature. After washing three times with TBST buffer, the membrane was visualized using a fluorescent Western blot imaging system (LI-COR Odyssey Clx).