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).