Anti β actin antibody ac 74

Recombinant M-CSF and RANKL were purchased from PeproTech. Recombinant mouse S100A4 was purchased from Prospec. Recombinant OPG was purchased from R&D Systems. Antibodies against vimentin and c-Fos were purchased from Santa Cruz. Antibodies against N-cadherin and snail2 were obtained from Cell Signaling Technology. Antibodies for RAGE (DD/A11) were from Millipore. Anti-S100A4 antibody was purchased from Abcam. Anti-β-catenin antibody was purchased from Invitrogen. Antibodies for NFATc1 (7A6), E-cadherin, and hHLA were purchased from BD Pharmingen. Anti-β-actin antibody (AC-74) and the leukocyte acid phosphatase kit (for TRAP staining) were purchased from Sigma-Aldrich. Lipofectamine for siRNA transfection was purchased from Life Technologies. siRNA oligonucleotides and shRNA lentiviral particles were purchased from Santa Cruz. Dentin slices were purchased from Immunodiagnostic Systems.

Protein sample from cultured mouse mammary tissue was prepared by homogenization in whole tissue extract buffer and then diluted by the same volume of 2×SDS sample buffer [37 ]. Equal amounts of protein (20 μg/lane) were resolved by SDS polyacrylamide gel (10%) electrophoresis and transferred to Immobilon-P nylon membrane (Millipore Co.). The membrane was blocked in Tris-buffered saline and Tween 20 (TBST: 10 mM Tris, pH 7.5; 150 mM NaCl; and 0.05% Tween 20), containing 5% (w/v) nonfat Carnation instant milk (Nestle, Solon, OH). Blot was incubated with primary antibody (1 : 10,000 of anti-β-actin antibody, AC74, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) overnight at 4°C. After washing in TBST, it was incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000 of donkey anti-mouse IgG, Sigma-Aldrich Chemical Co.) for 1 hour at room temperature. Then, it was visualized by using enhanced chemiluminescence Western blotting detection reagents (Amersham Pharmacia Biotech, Newark, NJ, USA) and appropriate exposure to X-ray film (Kodak, Rochester, NY, USA). Specific bands were quantified by densitometric analyses using a GelPro analyzer (Media Cybernetics, Bethesda, MD, USA).

Res2‐Igrov1 cells were grown to 80–90% subconfluency, trypsinized, and lysed in RIPA Lysis Buffer (Santa Cruz, Dallas, TX, USA). Subsequently, 20 µg whole cell lysate (per sample) was subjected to a NuPAGE 4–12% Bis‐Tris protein gel and transferred onto nitrocellulose (NC) membranes (Amersham™ Protran™ Premium 0.45 µm NC; GE Healthcare Life science, Chalfont St Giles, UK). Subsequently, (dissected) NC membranes were incubated with anti‐β‐actin antibody (AC‐74; Sigma‐Aldrich, Taufkirchen, Germany, 1 : 10⁶), anti‐cleaved PARP (Asp214) antibody (D64E10; Cell Signaling Technology, 1 : 250), or primary rabbit anti‐phospho‐γ‐H2AX antibody (#2577; Cell Signaling Technology, 1 : 250). Subsequently, membranes were incubated for detection with secondary antibodies, raised against rabbit and linked with HRP (#7074; Cell Signaling Technology) or raised against mouse and linked with HRP (order# 115‐035‐003; Dianova, Hamburg, Germany). Detection was performed with ECL Plus Western Blotting Detection reagent (GE Healthcare, order# RPN2232).

Anti clathrin heavy chain antibody (BF-06, Thermo Scientific Pierce, Rockford, IL) was used as the primary antibody in immunofluorescence assays. Mouse monoclonal antibodies to gB (2F12), pUL44 (ICP36), and IE1 (CH160) were purchased from Virusys corporation, Sykesville, MD, USA. Fluorescent label tagged secondary antibody DYLIGHT 594 was purchased from Thermo Scientific Pierce and used in immunofluorescent assays (IFA) described below. Hoechst 33258 (Thermo Scientific Pierce) staining identified the nuclei in IFA. Anti β-actin antibody (AC-74, Sigma-Aldrich, St Louis, Mo, USA) was used as a control for sample loading in immunoblots. Peroxidase-labeled horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) was used as the secondary antibody for IBs. Blots were detected using ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, United Kingdom).

Anti-human CD133 antibody (W6B3C1, 293C3) and mouse IgG were purchased from Miltenyi Biotec, anti-β-actin antibody (AC-74) was from Sigma Aldrich. Antibodies against cleaved Notch1 (Val1744) and CD133 (C24B9) were purchased from Cell Signaling Technology and anti-V5 antibody was from Bethyl Laboratories. Doxycycline (Dox) was from Clontech and G418 was from Roche Diagnostics.