Rabbit anti activated notch1

The cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested on ice. The protein concentration of the resulting lysates was determined using a RCDC protein assay kit (Sigma, USA). A prestained standard protein molecular weight marker and the protein samples were loaded into wells and separated using 8% SDS–PAGE. The blots were then transferred using electroblotting to polyvinylidene difluoride (PVDF) membranes. After the membranes were blocked with 5% nonfat dried milk or bovine serum albumin (BSA), the desired bands were incubated overnight at 4 °C with the corresponding primary antibodies, including rabbit anti-activated Notch1 (1:1000 dilution; Abcam, UK), rabbit anti-integrin β1 (1:1000 dilution; Abcam, UK), rabbit anti-HIF-1α (1:1000 dilution; Proteintech, USA) and mouse anti-β actin (1:1000 dilution; Santa Cruz, USA). Horseradish peroxidase-conjugated secondary IgG (1:5000 dilution; Proteintech, USA) was subsequently used as the secondary antibody. The results were analyzed using a ChemiDoc imaging system (Bio-Rad, USA).

Protein were obtained from cells lysed with radioimmunoprecipitation (RIPA) buffer supplemented with 1x complete protease inhibitor (Sigma-Aldrich) followed by sonication and centrifugation. SDS-PAGE of lysates was performed with the Bolt™ 4 to 12%, Bis-Tris system (Invitrogen) and protein transfer was conducted with the Biorad Trans-Blot Turbo Transfer System according to the manufacturer’s protocols. Immunoblots were labelled with following primary antibodies after blocking with Licor Intercept blocking buffer: rabbit anti-SOX9 (1:1000, Merck Millipore, Burlington, Massachuesetts, USA), rabbit anti-activated NOTCH1 (1:1000, Abcam) and mouse anti-β-actin (1:5000, Sigma-Aldrich). Primary antibodies were detected using fluorescently conjugated secondary antibodies: goat anti-rabbit IgG IRDye^® 800CW and goat anti-mouse IgG IRDye^® 680RN (1:2500, LI-COR Biosciences, Lincoln, Nebraska, USA). Detection and quantification of fluorescence intensity were performed using an Odyssey CLx imaging system (LI-COR Biosciences, Lincoln) and Odyssey 2.1 software or ChemiDoc MP Imaging System and ImageLab software v6.1. For an example of presentation of full scan blots, see Supplementary information.