P15ink4b

SOX2 (D6D9), OCT4 (C30A3), γH2AX (Ser139), CDK1, CDK2, p15INK4B, Cyclin D1 (DSS6), p-Aurora A (T288), p-Aurora B (T232, 1:1000), Aurora B, c-Myc (9E10) and Phospho-Chk2 (Thr68), Phospho-ATM (Ser198), Phospho p53 (Ser 15) antibodies were purchased from Cell Signalling Technology. GFP (clone B-2) antibody was purchased from Santa Cruz Biotechnology. Primary antibodies are used in 1:200 dilution for Immunofluorescence assay and 1:1000 dilution for Western blotting. GAPDH (clone MAB374, 1:4000) antibody was purchased from Millipore.
Horse radish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit secondary antibodies (1:5000) were purchased from Jackson ImmunoResearch. Alexa Fluor 488 goat anti-mouse antibody and Alexa Fluor 568 goat anti-rabbit antibody (1:200) were purchased from Life Technologies. Anti-PRL3 monoclonal antibody (mAb) (clone 318, 1:200 for Immunofluorescence and 1:2000 for western blot) was generated in-house. PRL3-zumab was engineered based on the original framework of murine anti-PRL3 mAb (clone 318).

Immunoblotting was conducted as previously described [43 (link)]. Membranes were probed with antibodies to p16^INK4a (PA5-20379, Pierce, Rockford, IL; USA), p15^INK4b (4822, Cell Signaling Technology (CST), Danvers, MA, USA), p18^INK4c (39-3400, Life Technologies, Carlsbad, CA, USA), p19^INK4d (PA5-26413, Pierce), p21^CIP/WAF (2946, CST), p27^KIP (2552, CST), CDK2 (sc-163, Santa Cruz Biotechnology (scbt), Santa Cruz, CA, USA), CDK4 (ab7955, Abcam, Cambridge, MA, USA), CDK6 (ABC275, Millipore, Temecula, CA, USA), Cyclin-A1 (sc-596, scbt), Cyclin-B1 (4135, CST), Cyclin D1 (ab134175, Abcam), Cyclin-E2 (4132, CST), GAPDH (G8795, Sigma), Tubulin (T5168, Sigma), STAT-3 (9132, CST) and pS727-STAT-3 (9134, CST), Tau (DAKO), pThr205-Tau (T6694, Sigma). Antibodies to Inhibitor-1 and pS6-inhibitor-1 were generated and characterized in-house [25 (link)]. The same membrane was probed for several proteins of different molecular weights after stripping in buffer containing 61 mM Tris-Base, 2%SDS and 7% β-mercaptoethanol at 50°C for 30 min. Immunoblots were quantified using Quantity One (BioRad). Samples were normalized to GAPDH (Figures 3 and 4) or to total protein levels as determined by Coomassie blue (CB) staining (Figure 5).

PTER of 98% purity was purchased from Enzo Life Sciences (Lausen, Switzerland). A 100 mM stock solution of PTER was made in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO) and stored at −20°C. The final concentration of DMSO for all treatments was <0.5%. Antibodies, specifically of cleaved caspase-3, caspase-8, caspase-9, poly(ADP-ribose) polymerase (PARP), heat shock protein 70 (HSP70), p-extracellular signal-regulated kinase (ERK)1/2, p-p38, p-c-Jun N-terminal kinase (JNK), ERK1/2, p38, JNK1/2, CDK 2, cathepsin B, C23, and β-actin (for the Western blot analysis), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against cyclin-dependent kinase (CDK)6, p21 Cip1, p27 Kip1, p15 INK4B, and cyclin D3 were purchased from Cell Signaling Technology (Danvers, MA). Anti-cyclin A2 and anti-cyclin E2 antibodies were purchased from Epitomic (Burlingame, CA). 4′-6-Diamidino-2-phenylindole (DAPI) was purchased from Sigma. The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB202190, the ERK1/2 inhibitor, U0126, and the JNK1/2 inhibitors, SP600125 and JNK-IN-8, were purchased from Calbiochem (San Diego, CA). The caspase-3 inhibitor, Z-DEVE-FMK, was purchased from BioVision (Mountain View, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma.