Rabbit monoclonal anti e cadherin

Western immunoblot analysis was performed using whole-cell lysates derived from HGSOC cell lines as previously described with minor alterations.^{15 (link)} To lyse cells grown as spheroids, 1% sodium dodecyl sulfate was added to the lysis buffer. Approximately 40 μg of protein lysate per cell line was analyzed. Equal loading was verified by incubating the membranes with anti-GAPDH antibody. Primary antibodies used include monoclonal mouse anti-GAPDH (GeneTex, Irvine, CA, USA; cat. no. GTX627408), monoclonal rabbit anti-E-cadherin (Cell Signaling, Danvers, MA, USA; cat. no. 3195), polyclonal rabbit anti-SUSD2 (Prestige Antibodies Sigma-Aldrich Corp.; cat. no. HPA004117) and polyclonal rabbit anti-N-cadherin (Cell Signaling; cat. no. 4061). Secondary antibodies used were Pierce (Thermo-Fisher Scientific, Waltham, MA, USA; cat. no. 31329) for GAPDH. Pierce cat. no. 31345 was used for N-cadherin, E-cadherin and SUSD2. Experiments were performed using three biological replicates for each cell line.

Using the BCA method to determine the protein concentration, 100 µg protein was added to 7.5% SDS-PAGE for analysis. Then, the protein was transferred to a PVDF membrane after electrophoresis, blocked in TBST solution containing 5% skimmed milk powder, and incubated with antibody. The protocols were performed as previously described (18 (link)). The monoclonal rabbit anti-ZNF326 (1:1000, ABclonal Technology, USA, A16477), monoclonal rabbit anti-E-cadherin (1:1000, Cell Signaling Technology California, USA, #3195), monoclonal rabbit anti-N-cadherin (1:1000, Cell Signaling Technology California, USA, #13116), monoclonal rabbit anti-Vimentin (1:1000, Cell Signaling Technology California, USA, #5741), monoclonal rabbit anti-AEG-1 (1:1000, Abcam, England, ab227981), monoclonal rabbit anti- NF-κB (1:1000, Cell Signaling Technology California, USA, #8242), monoclonal rabbit anti-ERCC1 (1:1000, Abcam, England, ab129267), and monoclonal mouse anti-β-actin antibody (1:2000; Beyotime Biotechnology, Nantong, China, AF5001) were used as primary antibodies. ImageJ software (version 1.44, Wayne Rasband, National Institutes of Health, USA) was applied to quantify the correction of these protein bands with the corresponding β-actin level. The fold change of figures was relative to β-actin.