2.12 | Western Blotting
Proteins from LF tissues and cells were extracted by Protein Extraction Sample Kit (Sigma, USA). Subsequently, proteins were separated by SDS-PAGE, converted to a nitrocellulose membrane (Sigma, USA) by electroblotting. The membranes were blocked for 2h at room temperature with 5 % skim milk (Sigma) and probed with primary antibodies against WISP-1 (Abcam), Smo (Abcam), Gli1 (Abcam), Shh (Abcam), collagen I (Abcam), collagen III (Abcam), and ɑ-SMA (Abcam,), and hybridized overnight at 4℃ with gentle shaking on the shaker. Finally, the membranes were incubated with 1:5000 goat anti-rabbit or anti-mouse secondary antibodies for 2h at room temperature (Abcam). GAPDH (Bioworld) was used as an internal control. The intensity of each blotting band was detected using a western blotting chemiluminescence kit (Bioworld).