Anti beta actin primary antibody

PCa cells seeded in 100-mm cell culture dishes were treated with Etn for different time points. After treatment, cells were subjected to immunoblotting as described previously 33 (link). Lysates from fresh tumor samples were prepared using BeadBlaster microtube homogenizer (LabRepCo) following the manufacturer's guidelines. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with anti-light chain 3 primary antibody (LC3; Cell Signaling Technologies, #2775S, 1:500) or GLUT1 primary antibody (Abcam, #ab115730, 1:1000) overnight at 4 °C; anti-beta-actin primary antibody (Santa Cruz Biotechnology, #SC-47778, 1:1000) was used as a loading control. Subsequently, membranes were incubated with goat anti-mouse (Santa Cruz Biotechnology, #sc-516102, 1;8000) or goat anti-rabbit (Santa Cruz Biotechnology, #sc-2357, 1:8000) IgG horseradish peroxidase-conjugated secondary antibodies. All antibodies against pro-apoptotic proteins were from Pro-Apoptosis Bcl-2 Family Antibody Sampler Kit (Cell Signaling Technologies, #9942T). The signal was visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, #32106). Signal intensities were quantified using ImageJ and were normalized to the respective β-actin signal intensity.

Western blots were executed using a WES instrument (Protein simple, CA, USA) according to the manufacturer’s guidelines. Briefly, cultured cells were lysed on ice using 1x RIPA buffer (Thermo Fischer, USA) followed by sonication and centrifugation steps to collect the protein supernatant. Protein concentrations in the supernatant were measured using BCA protein estimation assay kit (Thermo Fischer, USA). Protein samples (2 µg), wash buffers, blocking reagent, antibodies, and chemiluminescent substrate were prepared and distributed into the appropriate wells of assay plates. Subsequently, assay plates were loaded to the WES instrument, and proteins were allowed to separate. Detection of bands was performed automatically in the individual capillaries by the WES instrument. Anti HIF1A primary antibody (Novus biologicals, USA; Catalog# NBP1-02160SS; 1:250) and anti-beta actin primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA; Catalog No sc-47778: 1:250) were used together with anti-rabbit and anti-mouse secondary antibody supplied by ProteinSimple.

HPLC grade reference standards 3,4,5,-trihydroxybenzoic acid (gallic acid ethyl ester, purity 98%) was purchased from Acros Organics, Fischer Scientific; and 3,3′,4′,5,6-pentahydroxy flavones (quercetin dihydrate, purity 98%) was purchased from Fluka Biochemika. Urea and thiourea were purchased from Hi-Media Laboratories, India. 2,3,7,8-Tetrahydroxy-chromeno[5,4,3-cde]chromene-5,10-dione (ellagic acid), dithiothreitol (DTT), acrylamide, bis-acrylamide, ammonium persulfate, tetramethylethylenediamine (TEMED), sodium dodecyl sulphate (SDS), tris-HCL, phenylmethanesulfonylfluoride (PMSF), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), tween 20, and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate were purchased from Sigma-Aldrich, USA. Polyvinylidene fluoride (PVDF) membranes were purchased from Millipore (India) Pvt., Ltd., India. Mouse monoclonal anti-Bcl-2 primary antibody, anti-beta-actin primary antibody, rabbit polyclonal anti-Bax primary antibody, HRP conjugated anti-mouse IgG secondary antibody, HRP conjugated anti-rabbit IgG secondary antibody, and nonfat dry milk were purchased from Santa Cruz Biotechnology, CA. Methanol, glacial acetic acid, formaldehyde, hematoxylin and eosin stains, and other analytical chemicals and reagents used for HPLC and phytochemical analyses were purchased from Merck Pvt., Ltd., India.