Alkaline phosphatase linked antibodies for western blotting

Monoclonal antibodies to p65, and phospho-specific p65-NF-κB, MMP-9, CXCR4, cleaved-Caspase-3, and β1-Integrin, were obtained from R&D Systems (Heidelberg, Germany). Antibodies to β-actin were from Sigma-Aldrich (Taufkirchen, Germany). Secondary rhodamine-coupled antibodies for immunofluorescence and anti-Ki67 were from Dianova (Hamburg, Germany), and alkaline phosphatase-linked antibodies for Western blotting were from EMD Millipore (Schwalbach, Germany). Monoclonal anti-ALDH1 was obtained from Acris Antibodies GmbH (Herold, Germany). Monoclonal anti-CD133 and anti-CD44 were purchased from Abcam PLC (Cambridge, UK). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 4′,6-diamidino-2-phenylindole (DAPI), and alginate were from Sigma-Aldrich (Taufkirchen, Germany). TNF-β was purchased from eBiosciences (Frankfurt, Germany). Furthermore, TNF-β was given as a kind gift by Genetech (South San Francisco, CA, USA). Calebin A was a generous gift from Sabinsa Corporation (East Windsor, NJ, USA). Calebin A was diluted as 10,000 µM stock solution in dimethyl sulfoxide (DMSO) and further diluted in cell culture medium for experimental investigations. The final concentration of DMSO did not exceed 0.1% during the experiments.

Monoclonal antibodies to p65, as well as phospho-specific p65-NF-κB, MMP-9, CXCR4, cleaved-Caspase-3, β1-Integrin and PARP, were obtained from R&D Systems (Heidelberg, Germany). Antibodies to β-Actin were from Sigma-Aldrich (Taufkirchen, Germany). Secondary rhodamine-coupled antibodies for immunofluorescence and anti-Ki67 were from Dianova (Hamburg, Germany) and alkaline phosphatase-linked antibodies for Western blotting from EMD Millipore (Schwalbach, Germany). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DAPI, 5-Fluorouracil (5-FU), alginate, BMS-345541 and dithiothreitol (DTT) were from Sigma-Aldrich (Taufkirchen, Germany). Stock solutions of BMS-345541 (1000 µM) and of DTT (1000 mM) were prepared in PBS and further diluted in normal cell culture growth medium to obtain final concentrations. Then, 5-FU was diluted as a 1000 µM stock solution in ethanol and further diluted in the cell culture medium. Final concentrations of ethanol did not exceed 0.1% during treatment. TNF-β was purchased from eBiosciences (Frankfurt, Germany). Further, TNF-β was given as a kind gift by Genetech (South San Francisco, CA, USA). Calebin A (CA), with a purity of 99.65%, was a generous gift from Sabinsa Corporation (East Windsor, NJ, USA). Epon for electron microscopy was purchased from Plano (Marburg, Germany).

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.