Dnmt3a antibody

The newly added protease inhibitor and the lysis buffer (SDS) were applied for protein separation. Subsequent to separation through SDS-PAGE, the lysates were transferred onto a polyvinylidene fluoride membrane acquired from Roche. Then the membrane was blocked with 2% BSA, followed by incubation with anti-CD63 and anti-TSG 101 from Santa Cruz, and anti-Alix, ZEB1 antibody, MTOR antibody, DNMT3A antibody and GAPDH antibody from Abcam at 4°C overnight. Then the membrane underwent incubation with the proper secondary antibodies, and the protein expression in cells was measured with GAPDH as an internal reference.

As previously stated, LSCC cells were gathered and analysed using Western blot to evaluate the expression of DNMT3A.²⁴ Total protein was extracted using RIPA Lysis Buffer (Beyotime Biotechnology, China). BCA Protein Assay Kit (Beyotime Biotechnology, China) was used to measure the protein concentration. DNMT3A antibody (Abcam, Shanghai, China) was diluted to 1:1000. The expression levels of DNMT3A were expressed as a ratio of the expression of GAPDH.

RPTECs were cultured on the EZ slides (Merck Millipore). Treatment of Ang-II, AGE and HG was given to the cells and subsequent transfection with miR29b mimics and miR29b inhibitors was carried out. The cells were fixed using 4% paraformaldehyde. RPTECs based in vitro DN model was incubated with primary DNMT3A antibody (1:1000, Abcam), DNMT3B antibody (4 μg/ml, Sigma), DNMT1 antibody (1 μg/ml, Abcam) for overnight at 4 ºC. After overnight incubation with primary antibody, the slides were washed with PBS (3 times). The slides were then incubated with secondary antibody goat anti-rabbit IgG-FITC for 2 h. After complete incubation, slides were again washed with PBS (3 times) and counter stained with DAPI for cell nuclei. Slides were then observed under the microscope and images were quantified using Image J software.