Anti ptb

Cell lysates were prepared with Laemmli buffer supplemented with protease inhibitors (complete tablet; Roche) and phosphatase inhibitors (PhosSTOP tablet; Roche) and analysed by Western blotting with the following primary antibodies: anti-γH2AX (Abcam); anti-PTB (Abcam); anti-nPTB (Abcam); anti-βIII Tubulin (Abcam); anti-Tyrosine Hydroxylase (Abcam); anti-GAPDH (Thermo Scientific); anti-MAP2 (Novusbio); anti-DROSHA (Cell Signaling); anti-DICER (Sigma); anti-H2AX (Millipore). Primary antibodies were revealed with peroxidase-conjugated goat anti-mouse or anti-rabbit antibodies (Jackson Immunoresearch Laboratories) and enhanced chemiluminescence system (Super Signal West Pico Pierce or Super Signal West Dura Extended). βIII Tubulin antibody was revealed with alkaline phosphatase-conjugated goat anti-chicken antibody (Santa Cruz) with a colorimetric assay.

Total protein extracted from normal skin and keloid lesions were prepared using the Total Protein Extraction Kit (Applygen, Beijing, China). Protein concentrations were determined using a BCA Protein Assay kit (Solarbio, Beijing, China). Western blotting was carried out using the antibodies for anti-PTB (1:1500 dilution; Abcam, HK, China) and anti-β-actin (1:5000 dilution; Abcam, HK, China), which was used as an internal control for protein loading normalization.