P glycoprotein monoclonal antibody f4

Cells were planted on cover slips and incubated in 24-well plates overnight. Before fixing with pre-cold 4% paraformaldehyde for twenty minutes, cells were washed twice with PBS softly. And then cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. After blocking with BSA for 30 minutes, cells were incubated with P-Glycoprotein Monoclonal Antibody (F4), (1 : 100; Thermo Fisher scientific) for overnight at 4 °C, followed by incubation with secondary antibody conjugated with FITC for 2 hours at room temperature in the dark. DAPI was employed to stain the nuclei. Fluorescence images were pictured with a CKX41 microscope (Olympus, Tokyo, Japan).

Total protein of the cells was extracted with total protein extraction (Millipore, Bedford, MA, USA). Protein concentration was determined using the BCA protein assay kit. Cell lysates containing an equivalent amount of protein (10 μg) were electrophoresed through 12% SDS polyacrylamide gels, and subsequently transferred onto a nitrocellulose membrane (Sigma-Aldrich, Shanghai, China). Western blot analysis was performed with P-Glycoprotein Monoclonal Antibody (F4), (1 : 100; Thermo Fisher scientific), anti-IKB alpha (ab32518) (1 : 5000; Abcam), anti-IKB alpha (phospho S36) (ab133462), (1 : 50 000; Abcam), NFkB p50 Monoclonal Antibody (5D10D11) (1 : 1000; Thermo Fisher scientific), anti-NF-κB p65 (ab16502) (1 : 5000; Abcam), GAPDH was used as an internal control. Afterwards, membrane was washed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies. Protein bands were photoed with ChemiDoc system (Bio-Rad). The expression levels of these proteins were quantified with Image J software.