Rabbit polyclonal anti actin antibody

Human E-selectin/Fc, human ICAM-1/Fc and human CXCL12 were from R&D Systems (R&D Systems, Minneapolis, MN, USA); fluorescein isothiocyanate (FITC) goat secondary antibody to mouse was from Sigma (Sigma-Aldrich, St. Louis, MO, USA); KIM127 mouse monoclonal antibody was from American Type Culture Collection (ATCC, Rockville, MD, USA); 327C and A mouse monoclonal antibodies were kindly provided by dr. Kristine Kikly (Eli Lilly and Co., Indianapolis, IN, USA); Tyrphostin AG490 and rabbit polyclonal anti-actin antibody were from Sigma; rabbit monoclonal anti-JAK2 (D2E12), was from Cell Signaling Technology (Danvers, MA, USA); siRNAs (ON-TARGETplus SMARTpool) were from Thermo Fisher Scientific (Fremont, CA, USA).

A mouse monoclonal anti-cytokeratin 7 antibody, rabbit polyclonal anti-actin antibody and JAK2/STAT inhibitor (AG490) were obtained from Sigma-Aldrich (St. Louis, Mo., USA). Bromodeoxyuridine (BrdU) was obtained from Thermo Fisher Scientific (Waltham, MA). An anti-BrdU antibody was obtained from Dako (Tokyo, Japan). A rabbit polyclonal anti-vimentin (H-84) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A PI3K inhibitor (LY294002) and mitogen-activated protein kinase (MAPK) inhibitor (U0126) were purchased from Calbiochem-Novabiochem Corporation (San Diego, CA). A mouse monoclonal anti-β-catenin antibody was obtained from BD Transduction Laboratories (San Jose, CA). Rabbit polyclonal anti-lipolysis-stimulated receptor (LSR) and anti-tricellulin (TRIC) antibodies were obtained from Zymed Laboratories (San Francisco, CA). Alexa 488 (green)-conjugated anti-rabbit IgG and Alexa 594 (red)-conjugated anti-mouse IgG antibodies were purchased from Molecular Probes, Inc. (Eugene, OR). Recombinant human leptin and recombinant human adiponectin were obtained from PeproTech (Rocky Hill, NJ). Metformin was purchased from Wako (Tokyo, Japan). Berberine was obtained from Tokyo Chemical Industry, Inc. (Tokyo, Japan).

The plasmid encoding His-fused full length human PARP1 was a gift from John Pascal and described elsewhere [52 (link), 53 (link)]. The plasmid encoding the HA-fused full length human EZH2 plasmid (pCMVHA hEZH2) was a gift from Kristian Helin (Addgene plasmid # 24230) [54 (link)]. The plasmid encoding GST-fused full length human EZH2 (Pgex-EZH2) was a gift from Mien-Chie Hung (Addgene plasmid # 28060) [55 (link)]. All plasmids were verified by sequencing. Rabbit polyclonal anti-His (WB 1/200, Ip: 2.5 μgr/sample), rabbit anti-Mouse IgG HRP conjugated (WB: 1/5000; IP 2.5 μgr/sample), and mouse anti-Rabbit IgG HRP conjugated (WB: 1/5000, IP: 2.5 μgr/sample) were from Santa Cruz Biotechnology. Monoclonal mouse anti-HA (WB 1/1000) was from Origene. Monoclonal mouse anti-HA (IP 2.5 μgr/sample) and rabbit polyclonal anti-Histone H3 (WB 1/2000) were from Abcam. Rabbit polyclonal anti-PARP1 (WB 1/10000) and rabbit polyclonal anti-H3K27me3 (WB 1:2000) were from Active Motif. Rabbit polyclonal anti-PAR antibody (WB 1/1000) was from Trevigen. Mouse monoclonal anti-EZH2 antibody (WB 1/2000; IP 2.5 μgr/sample) was from BD Bioscience. Rabbit polyclonal anti-Actin antibody (WB 1/100) was from Sigma Aldrich. Anti-mono-ADP-ribose binding reagent and Anti-mono- and poly-ADP-ribose binding reagent (IP 5 μgr/sample) were from Millipore.

Rabbit polyclonal anti-tricellulin (TRIC), anti-claudin (CLDN)-1 and -2 antibodies and mouse monoclonal CLDN-2 and anti-occludin (OCLN) antibodies were obtained from Zymed Laboratories (San Francisco, CA, USA). Rabbit polyclonal anti-lipolysis-stimulated lipoprotein receptor (LSR) antibodies were from Novus Biologicals (Littleton, CO, USA). A mouse monoclonal anti-LSR antibody was obtained from Abnova (Taipei, Taiwan). A rabbit polyclonal anti-p63 antibody was obtained from Abcam (Cambridge, UK). Mouse monoclonal anti-acetylated tubulin and antiCK7 antibodies and a rabbit polyclonal anti-actin antibody were from Sigma-Aldrich (St. Louis, MO, USA). Alexa 488 (green)-conjugated anti-rabbit IgG and Alexa 594 (red)-conjugated anti-mouse IgG antibodies and Alexa 594 (red)-conjugated phalloidin were from Molecular Probes, Inc. (Eugene, OR, USA). HRP-conjugated polyclonal goat anti-rabbit IgG was from Dako A/S (Glostrup, Denmark). The ECL Western blotting system was from GE Healthcare UK, Ltd. (Buckinghamshire, UK). A rabbit polyclonal anti-actin antibody and EGFR inhibitor (AG1478) were obtained from Sigma-Aldrich (St. Louis, MO, USA). A TGF-β receptor type 1 inhibitor (EW-7197) was obtained from Cayman Chemical (Ann Arbor, MI, USA). FITC-dextran (FD-4, MW 4.0 kDa) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA).

Astrocyte cultures were washed with PBS, fixed in paraformaldehyde 4% for 20 min at room temperature, washed in PBS (0.1 m) and blocked 1 h in PBS containing albumin (4%). The following primary antibodies were used: mouse anti-glial fibrillary acidic protein monoclonal antibody (dilution 1:3 000; Merck Millipore, Billerica, MA, USA; MAB3402), chicken anti-MAP-2 polyclonal antibody (dilution 1:3 000; Abcam, Cambridge, UK; ab5392), rabbit anti-clathrin heavy-chain polyclonal antibody (1 μg ml⁻¹; Abcam; ab21679), rabbit anti-actin polyclonal antibody (dilution 1:200; Sigma-Aldrich; A2066) and mouse anti-actin monoclonal antibody (dilution 1:1 000). Secondary fluorescent antibodies (dilution 1:600) were purchased from Jackson Immunoresearch (Bar Harbor, ME, USA). A counterstaining with DAPI (3 ng ml⁻¹) was realized in some experiments. Confocal laser-scanning microscopy was performed using a Leica SP5 II confocal microscope (Leica, Wetzlar, Germany).

Cell lysates were prepared using cell lysis buffer (1% NP-40, 0.5% sodium deoxycholic acid) supplemented with a protease inhibitor cocktail (Roche, Nutley, NJ). For detection of BIRC6 (528 kDa), 10 μg whole cell lysate was resolved in 5% SDS-polyacrylamide gel and electrotransferred to a PVDF membrane in tris (25 mM), glycine (191.5 mM), methanol (10%), SDS (0.05%) buffer at 40V overnight at 4°C. Membranes were probed with anti-BIRC6 antibody (1:500; Novus Biologicals) at room temperature for 2.5 hours. For detection of cIAP1 and survivin, lysate was resolved in 10% and 15% SDS-polyacrylamide gel, respectively, and electrotransferred to a PVDF membrane in tris (25 mM), glycine (191.5 mM), methanol (10%) buffer at 100V for 1 hour. Membranes were probed with anti-cIAP1 (1:500; Cell Signaling, #7065) and anti-survivin (1:500; Cell Signaling, #2808) antibodies at room temperature for 2.5 hours. Actin or vinculin were used as loading controls and detected on membranes using rabbit anti-actin polyclonal antibody (1:2000; Sigma-Aldrich) or mouse anti-vinculin antibody (1:5000; Sigma-Aldrich).