P65 nf κb

Monoclonal antibodies to phospho-specific-FAK, PARP, p65-NF-κB, phospho-specific p65-NF-κB, and activated-Caspase-3 were from R&D Systems (Heidelberg, Germany). Antibodies to β-Actin, cytochalasin D (CD), MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], DAPI (4,6-diamidino-2-phenylindole), dithiothreitol (DTT) and alginate were from Sigma-Aldrich (Taufkirchen, Germany). Anti-E-cadherin, anti-vimentin, anti-TGF-β1, anti-p-Smad2 and anti-Slug were from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Secondary antibodies for immunofluorescence were procured from Dianova (Hamburg, Germany). Alkaline phosphatase-linked antibodies for Western blotting were obtained from EMD Millipore (Schwalbach, Germany). Calebin A (CA), was a generous gift from Sabinsa Corporation (East Windsor, NJ, United States). Calebin A was diluted and 10,000 µM stock solution was prepared in DMSO, and this was further diluted in cell culture medium. Final concentration of DMSO did not exceed 0.1% during the experiments. Focal adhesion kinase inhibitor (FAK-I) (PF-562271) was purchased from Sellekchem (Munich, Germany). Stock sample of 10 mM FAK-I was prepared in DMSO and diluted again in serum-starved medium to make working samples.

The protein expression levels of target genes were detected by western blot. Total proteins were extracted from heart tissues or cultured cells using RIPA lysis buffer (Beyotime) according to instructions provided with the kit and protein concentrations were measured using a BCA Protein Assay Kit (Pierce Biotechnology Inc.). Antibodies used in this study are as follows: glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (Immunoway), p65NF‐κB (R&D Systems), NLRP3 (Cell Signaling Technology), and IL‐38 (R&D Systems). Equal amounts of denatured protein samples were separated on 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis prepared in advance for about 2 h and then were transferred to polyvinylidene difluoride membranes, followed by blocking with 5% nonfat milk for 2 h and incubated with the indicated primary antibodies at 4°C overnight. After incubation with horseradish peroxidase‐conjugated secondary antibodies for about 2 h at room temperature, membranes were treated with ECL reagents (Thermo Scientific). GAPDH was used as the loading control to normalize comparisons, and data were analyzed using a densitometer.