Rabbit anti cldnd1

Cells in 6-well cell culture plate were lysed using 150 μL RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors. Then cell lysates were collected and centrifuged at 10,000 rcf and 4℃ for 15 minutes. The protein concentrations were measured using the Modified BCA Protein Assay Kit (Sangon Biotech). After mixing and boiling with the SDS-PAGE loading buffer, 40 μg of total protein was electrophoresed in 10% SDS-PAGE gels and transferred on to a PVDF membrane (Merck Millipore). Then the PVDF membrane was blocked in PBST buffer supplemented with 5% skim milk and 0.1% Tween 20 for 1 hour at room temperature and incubated with the primary antibodies overnight at 4℃. After incubation with the consistent HRP-conjugated secondary antibodies, the protein bands were detected using SuperSignal^® West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The following antibodies were used according to the manufacturers' instructions: rabbit anti-CLDND1 (Abcam, MA, USA), rabbit anti-Flag (Sigma-Aldrich), mouse anti-β-actin (CMCTAG, WI, USA), HRP-labeled anti-rabbit IgG (KPL, MD, USA), and HRP-labeled anti-mouse IgG (KPL).

When the coverage of cells on cover slips reached about 90%, cells were fixed for 15 minutes at room temperature in 4% paraformaldehyde. After aspiration of fixative, samples were rinsed three times in 1×PBS for 5 minutes each and blocked in Blocking Buffer (1×PBS supplemented with 0.3% Triton X-100 (Sangon Biotech) and 5% normal goat serum (Life Technologies)) for 60 minutes. After incubation with the primary antibody (1:100) overnight at 4℃, samples were rinsed three times in 1×PBS for 5 minutes each and incubated with the anti-rabbit IgG second antibody (1:500) for 60 minutes at room temperature in dark. The normal rabbit IgG (Life Technologies) was used as negative control. The antibodies for immunofluorescence were as follows: rabbit anti-CLDND1 (Abcam), rabbit anti-IgG (Merck Millipore), and anti-rabbit IgG (Alexa Fluor^® 488 Conjugate) (Life Technologies). The nuclei was labeled using Hoechst 33258 (Sangon Biotech), and cells were visualized using a fluorescence microscope Ti-S (Nikon, Tokyo, Japan).