Mouse anti β actin monoclonal antibody

The cells were lysed in buffer containing 50 mM Tris-HCl (pH 6.9; Sigma), 2% sodium dodecylsulfate (SDS; Nacalai), 6% 2-mercaptoethanol (Sigma), and 10% glycerol. Protein samples (20 μg) were subjected to 4%–20% SDS-polyacrylamide gel electrophoresis and subsequently transferred onto an Immuno-Blot PVDF membrane (Bio-Rad). The membrane was treated with a mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology) at a dilution of 1 : 1000 or goat polyclonal anti-MEST antibody (Abcam) at a dilution of 1 : 200, followed by biotinylated anti-mouse IgG (Nichirei Biosciences, Inc., Tokyo, Japan) or anti-goat IgG (Nichirei) as appropriate. After the membrane was washed thoroughly, the reactive bands were visualized using the ECL Select western blotting detection system (GE healthcare, Buckinghamshire, UK) and Image Quant LAS 500 (GE).

We acquired a rabbit polyclonal anti-CNX (C-terminal) antibody (Cat No. SPA-860), rabbit polyclonal anti-CRT antibody (Cat No. SPA-600), rabbit polyclonal anti-BiP antibody (Cat No. SPA-826), and mouse monoclonal anti-PDI antibody (Cat No. SPA-891) from Stressgen (San Diego, CA, USA). A mouse monoclonal anti-β-actin antibody was acquired from Santa Cruz Biotechnology (Cat No. sc-1616, Dallas, TX, USA). We obtained a rabbit polyclonal anti-CNX (N-terminal) antibody (Cat No. ADI-SPA-865), and mouse monoclonal anti-E-cadherin antibody (Cat No. 610181) from Enzo Life Sciences (New York, NY, USA), and BD Biosciences (Franklin Lakes, NJ, USA), respectively. Rabbit polyclonal and mouse monoclonal anti-β-hCG antibodies were purchased from Proteintech (Cat No. 11615-1-AP, Rosemont, IL, USA) and Abcam (Cat No. ab9582, Cambridge, UK), respectively. Rabbit polyclonal and mouse monoclonal anti-luteinizing hormone/chorionic gonadotropin receptor (LHCGR) antibodies were obtained from Thermo Fisher Scientific (Cat No. PA5-97923, Waltham, MA, USA) and Novus Biologicals (Cat No. NBP2-53726, Centennial, CO, USA), respectively. Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Dako (Cat No. P0448 and P0260, Glostrup, Denmark). All other reagents used in this study were of high grade and were obtained from Sigma-Aldrich and Wako Pure Chemicals (Osaka, Japan).

Bicinchoninic acid (BCA) protein assay kit (P0009), LDH cytotoxicity assay kit (C0016), enhanced chemiluminescence (ECL) kit (P0018M), MTT cell proliferation and cytotoxicity assay kit (C0009), Hoechst staining kit (C0003), benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD) (C1202), were obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Glutathione (GSH) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Jiancheng, Nanjing, Jiangsu, China). Hydrogen peroxide (H₂O₂) and 3-methyladenine (3-MA) were obtained from Sigma-Aldrich (St. Louis, USA). Rabbit monoclonal anti-caspase-3 (cleaved) antibody was obtained from Beyotime Institute of Biotechnology. Rabbit polyclonal anti-LC3B antibody and horseradish peroxidase (HRP)-conjugated goat anti-rabbit or -mouse secondary antibodies were purchased from Sigma-Aldrich. Mouse monoclonal anti-β-actin antibody and rabbit polyclonal anti-ATG5 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). X-tremeGENE siRNA transfection regent was from Roche (Basel, Switzerland).

Rabbit polyclonal anti-Kif2a antibody used in Western blot and immunofluorescence was purchased from Novus Biologicals (Littleton,CO); Mouse monoclonal anti-α-tubulin-FITC antibody and mouse monoclonal anti-γ-tubulin antibody were obtained from Sigma-Aldrich Co. (Cat# F2168, Ca#T6557); Rabbit polyclonal anti-bub3 antibody was obtained from Santa Cruz Biotechology (Ca#sc-28258); Human polyclonal anti-centromere antibody (ACA) was obtained from Antibodies Incorporated (Item # 15-234-0001). Alexa Fluor@ 488-conjugate Goat anti-Rabbit IgG (H + L) and Alexa Fluor @594-conjugate Goat anti-Rabbit IgG (H + L) were produced by Thermo Fisher Scientific (Catalog# A-11008, Catalog# A-11012); Cy5-conjugated goat anti-human IgG and Cy5-conjugated goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA) (Cat#115-175-146, Cat#115-175-062). Mouse monoclonal anti-β-actin antibody was purchased from Santa Cruz Biotechnology (SC-8432, Santa Cruz, CA).
All other reagents were purchased from Sigma Aldrich except when mentioned otherwise.

Cells were treated for 24 h with ART. Cells were then changed with fresh medium and irradiated at the indicated doses. The cells were then washed twice with ice-cold PBS and directly lysed in 200 μl of cell lysis buffer. Tumors from nude mice were resected, homogenized and lysed. Western blot was performed as described previously [13 (link)]. Primary antibodies against Wee1, Cyclin B1, P53 and Cdc2 were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a 1:1,000 -1:2,000 dilution. β-Actin was used as the loading control and detected using a mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology).