Mouse monoclonal anti clc 3 antibody

The total protein levels of the fresh cervical tissue specimens (N = 45, SCC = 60, PN = 60) and cultured cells were evaluated by using the BCA assay kit (SinoBio Biotech) to measure protein concentration which was performed following the manufacturer’s instructions. Equal amounts of protein were analysed by electrophoresis in 8% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). The membranes were blocked using 5% skim milk for 2 h at room temperature. Subsequently, the membranes were incubated with the mouse monoclonal anti-ClC-3 antibody (diluted 1:1000, Abcam) or the mouse anti-ß-actin antibody (diluted 1:1000, Beyotime Biotechnology Inc.) overnight at 4 °C. The membranes were then incubated in the HRP-conjugated secondary antibody for 2 h at room temperature. The proteins were measured by using the ECL system (CWBIOTECH, China) with the ChemiDoc XRS system (Bio-Rad, Philadelphia, USA).

Cells were grown on 6 mm round coverslips, then washed with PBS, and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then rinsed with PBS and treated with 0.5% Triton X-100 for 5 min to permeabilize followed by blocking with 10% sheep serum for 45 min. Afterward, the cells were incubated with primary antibodies (rabbit anti-ClC-3 antibody, Cell Signaling Technology; mouse monoclonal anti-ClC-3 antibody, Abcam; rabbit anti-ERα antibody and rabbit anti-ERβ antibody, Abcam) at 4°C overnight. The cells were washed with 1% sheep serum and incubated with secondary antibody (Alexa Fluor 488-labeled goat anti-rabbit IgG, Cy3-labeled Goat Anti-Rabbit IgG (H + L), Alexa Fluor 488-labeled Goat Anti-Mouse IgG(H + L), Beyotime Institute of Biotechnology, China) for 1 h. The cells were rinsed with PBS and exposed to Hoechst 33,258 for 5 min to stain the nuclei. The cell side of the coverslips was attached to the mounting medium on glass slides. Immunofluorescence was detected under a Nikon C1-si confocal microscope (Japan).