Western blot analysis was performed on whole cell extracts (30 µg lysate) of DU 145 (Lane 1), MDA-MB-231 (Lane 2), U87 MG (Lane 3), SH-SY5Y (Lane 4), HeLa (Lane 5) and A-431 (Lane 6). The blots were probed with Anti-NUMB Recombinant Rabbit Monoclonal Antibody (Product # 703078, 1:500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4,000 dilution). A 70 kDa band corresponding to NUMB was observed across the cell lines tested.
Knockdown of NUMB was achieved by transfecting PC-3 cells with NUMB specific siRNA (Silencer® select Product # s16466, s16465). Western blot analysis (Fig a) was performed using whole cell extracts from NUMB knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-NUMB Recombinant Rabbit Monoclonal Antibody (Product # 703078, 1:500 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA-mediated knockdown confirms that antibody is specific to NUMB.
Supplier Page from Thermo Fisher Scientific for NUMB Antibody