Anti notch1

Protein extraction and immunoblotting were performed as previously described [47 (link)]. Primary antibodies were as follows: anti-Notch1 (Clone A6 Novus Biologicals, Cambridge, UK), anti-Thbs1(sc-73158, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-E-Cadherin (Clone NCH-38, Dako, Denmark) anti-mTor (2993, Cell Signaling Technology, Beverly, MA), anti-Icam5 (Abcam, Cambridge, UK), anti Pai3 (sc-99153, Santa Cruz Biotechnology), anti-Vimentin (Clone V9, Dako), anti Ck19 (Clone RCK108, Dako), anti-Ck18 (Clone DC10, Dako), anti-Mmp-9 (Clone 6-6B, Calbiochem, San Diego, USA), anti-Snail (sc-28199, Santa Cruz Biotechnology), anti-Ck8 (sc-52324, Santa Cruz Biotechnology), anti-Alpha-Sma (Clone 1A4, Sigma), anti-E-Cadherin (Clone 4A2, Cell Signaling) and anti-β-Actin monoclonal antibody (Clone AC-40). Immunoreactivities were revealed with the EnVision dextran polymer visualization system (Dako). Membranes were washed and autoradiographies were obtained using a chemiluminescence reaction (ECL reagents, Amersham). Digital images of autoradiographies were acquired with a scanner (Fluor-S MultiImager, Bio-Rad) and signals were acquired in the linear range of the scanner and quantified using specific densitometric software (QUANTITY-ONE, Bio-Rad) in absorbance units.

Western blotting was used to confirm the protein level of those differentially-expressed genes of distal nerve segment that were identified from the PCR array of those C57BL/6 mice after nerve crush injury. The distal nerve specimens of 6 C57BL/6 mice specific for post-crush days 3, 7, and 14 were harvested and homogenized with tissue protein extraction reagent T-PER^TM (Pierce, Waltham, MA) containing phosphatase and protease inhibitors. The protein samples in 30 μg were resolved on 10% SDS-polyacrylamide gels and transferred to polyvinylidenedifluoride membranes. Blots were blocked with 5% skim milk in Tween-20/ phosphate-buffered saline, and incubated with various primary antibodies, i.e., rabbit anti–Sox2 (Millipore Biotechnology, Billerica, MA), anti–c–Jun (Millipore Biotechnology), anti-Notch1 (Novus Biologicals, Littleton, CO), anti-Mbp (Novus Biologicals), and anti-β-actin (Millipore Biotechnology), at 4°C overnight. The blots were then incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 60 min, and developed with ECL™ Western Blotting Systems (Amersham Pharmacia Biotech, Little Chalfont, UK). The protein bands were detected using a FluorChem 8900 imaging system and quantified with the AlphaEaseFC software (Alpha Innotech Corp, Santa Clara, CA).