β actin c 4 mouse monoclonal igg1

After sample preparation, protein lysates were obtained by lysing cells with RIPA Buffer (Thermo Scientific). Next protein level were quantitated by using Pierce Bicinchoninic Acid (BCA) PROTEIN Assay Kit (Thermo scientific) according to the manufacturer’s protocol. Based on the concentration measurements, equal amounts of protein were taken from each sample and prepared for standard SDS-PAGE polyacrylamide gel electrophoresis and western blotting, based on Bio-Rad Western Blotting Protocols (Bio-Rad). The membranes were imaged with a Li-COR Odyssey Infrared Imaging System to evaluate the protein expression. For western blotting, the primary antibodies used in this study were: HES5 (EPR15578) rabbit, NOTCH1/N1-TMICD (D1E11 XP(R)) rabbit mAb, NOTCH3/N3-TMICD (D11B8) rabbit mAb, HES1 (D6P2U) rabbit, JAG1 ((D4Y1R) XPCR) rabbit mAb. HES5 Ab was purchased from Abcam, (Cambrige, MA, USA), all other primary antibodies were purchased from Cell Signalling (Beverly, MA, USA). β-actin (C4) mouse monoclonal IgG1 (Santa Cruz Biotechnology) was used as house-keeping gene. The secondary antibodies used were anti-rabbit-IR800 (Li-Cor Biosciences, Lincoln, NE) and anti-mouse IgG (H + L) (DyLight 680 conjugate) (Cell Signalling Technology).