Rabbit anti human ilk monoclonal antibody

Western blot assays were performed as previously described. Cells were lysed (Bio-Rad), and the membranes were probed with rabbit anti-human-ILK monoclonal antibody (Abcam), rabbit anti-α-SMA monoclonal antibody (Abcam), rabbit anti-AKT monoclonal antibody (Abcam), rabbit anti-p-AKT monoclonal antibody (Abcam), and rabbit anti-GAPDH monoclonal antibody (Abcam) as an internal reference overnight at 4 °C. Membranes were washed in RIPA lysis buffer (Beyotime, CHN) and centrifuged at 12 000 g and 4 °C for 5 min to extract the total protein. The total protein concentration was measured using the bicinchoninic acid assay kit (Beyotime, Cnina) according to the manufacturer's instructions. We separated 20 nl of total protein in each sample hole using 10% SDS-PAGE and transferred to a nitrocellulose membrane. The proteins were then washed with TBST and incubated with horseradish peroxidase-labeled goat anti-rabbit secondary antibody (Beyotime, CHN) for 1 h at 37 °C, and incubated with enhanced chemiluminescence (Thermo, USA) for 5 min in the dark at room temperature. The photographs were collected using a gel imaging system (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).

Cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and washed. They were incubated with rabbit anti-human-ILK monoclonal antibody (Abcam) overnight at 4 °C followed by CY3-labeled anti-rat secondary antibodies (Abcam). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) and image acquisition was performed by laser confocal microscopy (Carl Zeiss, Jena, Germany).