Rabbit anti notch3

Melanoma cells were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Thermo Scientific) and protein concentration was obtained using Bio-Rad protein assay dye reagent concentrate (Bio-Rad, Hercules, CA). Equal amounts of proteins were loaded on 4–15% SDS-PAGE gel (Bio-RAD). SDS PAGE was run at 55mA for 50 min and proteins were transferred on nitrocellulose membrane (Bio-RAD). The blots were blocked for 45 min in 5% non-fat dry milk and probed using rabbit anti-Notch3 (Santa Cruz) or rabbit anti-ITGB1 (Abcam, 553715). Mouse anti-β-actin (Sigma) antibody was used as loading control. Primary antibodies were incubated 1 hr at room temperature followed by incubation in HRP-conjugated secondary antibody for 1 hr at room temperature (goat-anti mouse IgG and goat anti-rabbit IgG, Millipore, Burlington, MA). Bands were detected by chemiluminescence using ECL solution (WesternBright Sirius, Advansta, Menlo Park, CA) and visualized by Chemi-Doc (Bio-Rad).

The cells were washed twice with ice-cold PBS and lysed in RIPA buffer. The protein content of the lysate was determined using a BCA protein assay kit (Catalogue Number: P0012, Beyotime, Shanghai, China). The proteins were separated by 10% SDS polyacrylamide gels and transferred onto polyvinyl difluoride membranes. The membranes were blocked in 5% BSA in TBST for 1 hour at room temperature and then incubated with a primary antibody overnight at 4°C. The primary antibodies used were mouse anti-Oct-4 (Catalogue Number: A7920, 1 : 1000, ABclonal Technology), rabbit anti-Sox-2 (Catalogue Number: 27015, 1 : 1000, AbSci, USA), rabbit anti-Nanog (Catalogue Number: A3232, 1 : 1000, ABclonal Technology), anti-PPAR-γ (Catalogue Number: 2443, 1 : 1000, Cell Signaling, USA), rabbit anti-Notch3 (Catalogue Number: sc-515617, 1 : 1000, Santa Cruz, USA), rabbit anti-c/EBPα (Catalogue Number: 2295, 1 : 1000, Cell Signaling, USA), and mouse anti-β-actin (Catalogue Number: AB21800, 1 : 5000, AbSci, USA). The membranes were incubated with HRP-conjugated secondary anti-mouse or anti-rabbit antibodies and visualized via an enhanced chemiluminescence (ECL) kit (Catalogue Number: P0018S, Beyotime Biotechnology, Shanghai, China) using ChemiDoc XRS with Quantity One Software (Bio-Rad, Hercules, CA). Bands on immunoblots were analyzed by densitometry.