Anti p21 12d1

Cells were lysed in 2% SDS buffer (25 mM Tris–HCl pH 7.5, 100 mM Nacl, 3 mM EDTA, 7% Glycerol) and fresh protease inhibitors. Extracts were sonicated for 10 s and centrifuged at 12000 × rpm for 10 min to remove cell debris. Western blotting was performed using the following primary antibodies: mouse monoclonal anti-Gapdh 6C5 (#sc32233, Santa Cruz Biotechnology, Heidelberg Germany); mouse monoclonal anti-b-Actin (ACTBD11B7) (#sc-81178, Santa Cruz Biotechnology, Heidelberg Germany); mouse monoclonal anti-Tubulin B512 (#T5168, Sigma-Aldrich, Darmstadt, Germany); rabbit polyclonal anti-Golm1 (#PA5-30622, Thermo Fisher Scientific, Waltham, MA USA); rabbit monoclonal anti-Synaptophysin (#ab32127, Abcam, Cambridge, UK); mouse polyclonal anti-Psat1 (#PA5-22124, Thermo Fisher Scientific, Waltham, MA USA), rabbit polyclonal anti-CDH2 (#ab18203, Abcam, Cambridge, UK), rabbit monoclonal anti-EGFR (#C74B9, Cell Signaling, Danvers, MA, USA) and rabbit polyclonal anti-p21 (12D1) (#2947 Cell Signaling, Danvers, MA, USA). As secondary antibodies goat anti-mouse and anti-rabbit conjugated to horseradish peroxidase were used (Bethyl Laboratories, Montgomery, TX, USA). Western blot analysis was performed with the aid of the enhanced chemiluminescence system (Thermo Fisher Scientific, Waltham, MA USA). ECL detection was done using a ChemiDoc-It Imaging System (UVP, Upland, CA) instrument.

The following rat anti-mouse mAbs; anti-CD11b (M1/70, TONBO Biosciences) and anti-CD19 (1D3, TONBO Biosciences), and mouse or rabbit anti-human mAbs; anti-CD38 (HB7, TONBO Biosciences), anti-p21 (12D1, Cell Signaling Technology), anti-AIM2 (3B10, BioLegend), anti-SKP2 [EPR3305(2), Abcam], and anti-USP10 (D7A5, Cell Signaling Technology) were used. Isotype-matched control IgGs for individual rat and mouse mAb were purchased from BD Biosciences. PE-conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific) was used as the secondary Ab for the flow cytometry analysis.

Nocodazole (Cat#487928) and Thymidine (Cat#89270) were purchased from Sigma-Aldrich LLC. Doxorubicin hydrochloride (Cat#2252) was obtained from R&D Systems, Inc. Irinotecan (Cat#S2217), oxaliplatin (Cat#S1224), and p53 activator nutlin3a (Cat#S8059) were all from Selleck Chemicals. Monoclonal antibody to PLK1 was purchased from Thermo Fisher Scientific Company. Antibody against CDC14B (Cat#sc-374572), cdc2 p34 (Cat#sc-54) and chk1 (Cat#sc-8408) were from Santa Cruz Biotechnology. Anti-p53 (Cat#2524), anti-p21 (12D1) (Cat#2947), anti-phospho-cdc2 (Tyr15) (Cat#9111), anti-phospho-cdc2 (Thr161) (Cat#9114), anti-Claspin (Cat#2800), anti-p-chk1 (ser-317) (Cat#8191), and anti-p-histone H3 (ser-10) (Cat#9701) antibodies were all from Cell Signaling Technology. CDC25A (Cat#ab989) and CDH1 (Cat#ab217038) were purchased from Abcam. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Trypsin (0.25%), and Penicillin- Streptomycin- Glutamine (100×) were obtained from Thermo Fisher Scientific company.