Mouse monoclonal anti numb

Cells were grown and differentiated on coverslips coated with 200 μg/ml poly-l-ornithine. Cells were then fixed in 4% formaldehyde for 20 minutes and stored in PBS. Membranes were permeabilized with 0.25% Triton X-100, and nonspecific binding was blocked with 1% BSA for 30 minutes. Cells were incubated overnight with primary antibodies: rabbit monoclonal anti-Notch2 (1:1600, CST, Shanghai, China), mouse monoclonal anti-Numb (1:1000, abcam, Shanghai, China). Cells were then incubated for 2 hours with goat anti-mouse or rabbit CY3 554–conjugated secondary antibodies (1:400). coverslips were then incubated with DAPI nucleic acid stain (l μg/ml) for 10 minutes and mounted with glycerin.

The paraffin embedded sections (thickness, 4μm) were de paraffinized completely. To retrieve the antigens, the slides were immersed in citric acid buffer (10 mmol/L of citrate sodium and 10 mmol/L of citric acid) and boiled in a microwave oven at 92–98°C for 15 minutes. The sections were cooled to room temperature and sequentially incubated at room temperature with 3% H2O2 in methanol for 15 minutes to quench endogenous peroxidase and in normal blocking serum for 30 minutes. The slides were then incubated with mouse monoclonal anti-XIAP (1:300, abcam, Shanghai, China), rabbit monoclonal anti-Survivin (1:300, abcam, Shanghai, China), mouse monoclonal anti-TTF-1 (1:500, abcam, Shanghai, China), mouse monoclonal anti-P63 (1:500, Santa Cruz, Shanghai, China), mouse monoclonal anti-CK5/6 (1:500, Santa Cruz, Shanghai, China), rabbit monoclonal anti-Notch1 (1:200, CST, Shanghai, China), rabbit monoclonal anti-Notch2 (1:200, CST, Shanghai, China) or mouse monoclonal anti-Numb (1:250, abcam, Shanghai, China) at 4°C overnight and stained with DAB. Intervening PBS washes were performed after incubation when necessary.

The cytoplasmic, membrane and organelle and cytoskeletal and nuclear fractions of cells were obtained by lysing using the Cell Fractionation Kit (#9038, Cell Signaling Technology). Whole‐cell proteins were extracted by lysing in RIPA buffer. The protein lysate was separated using 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and electro‐transferred onto a polyvinylidene fluoride membrane. The membranes were blocked in 5% BSA and were incubated with primary antibodies overnight at 4°C. The following antibodies were used: mouse monoclonal anti‐NUMB (1:1000, ab14140; Abcam), mouse monoclonal anti‐MDM2 [2A10] (1:50, ab16895; Abcam, Cambridge, MA, USA), Rabbit Polyclonal anti‐P53 (1:1000, #9282; Cell Signaling Technology), Rabbit Monoclonal anti‐Histone H3 (D1H2) (1:2000, #4499; Cell Signaling Technology), Rabbit Polyclonal anti‐Caveolin‐1 (1:1000, bs‐1453R; Bioss) and mouse anti‐GAPDH (1:1000, 60 004‐1‐Ig; Proteintech Group). The membranes were washed and incubated with anti‐rabbit or anti‐mouse secondary antibody (1:5000, SSA004/SSA007; Sino Biological Inc) for 2 hours at room temperature. Finally, antigen‐antibody complexes were detected using an electrochemiluminescence Western blotting detection reagent.