Cdk2 sc 163

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).

To analyze protein expression in cells, immunoblotting analysis was performed as previously described [2 (link)]. Antibodies against the following proteins were obtained from Santa Cruz Technology (California, US): Aur A (sc-25425), Aur B (sc-25426), BRCA1 (sc-6954), CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), cyclin D1 (sc-718), cyclin E (sc-247), cyclin A (SC-751). Antibodies against BRCA2 (19791-1-AP) and cyclin B1 (cs-4135) were obtained from Proteintech (Chicago, USA) and Cell Signaling Technology (Massachusetts, US), respectively. The detection of β-actin (A2228, Sigma Aldrich, St. Louis, MO) was used as a loading control. The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or of donkey anti-rabbit immunoglobulin linked to horseradish peroxidase from Cell Signaling Technology (Massachusetts, US). Immunoblotting reagents were from an electrochemiluminescence kit (Amersham Biosciences). To test whether the expression levels of proteins were regulated through proteasome-mediated degradation, cells were exposed with 20 μM MG132 (#S2619, Selleck Company, Texas, America) for 3 h and then harvested for Western blotting analysis.

Antibodies against NLRP3 (HPA012878), Tom20 (WH0009804M1), Flag (F1804), XRCC4 (UM500021), and Gprc 5a (HPA007928) were purchased from Sigma-Aldrich (St. Louis, MO, USA). pCHK2 (Tyr68, 2661S), GAPDH (5174P) and γH2AX Ser139(2577 L) were from Cell signaling (Beverly, MA). Caspase-1 (SC-56036), IL-1β (SC-52012), ASC (SC-271054), DNA PKcs (SC-390698), LigIV (SC-271299), CC-16 (SC-365992), and CDK2 (SC-163) were from Santa Cruz (Santa Cruz, CA, USA). Mito TEMPO, carbonyl cyanide 4-(trifluoromethoxy)-phenyl-hydrazone (FCCP), Antimycin A, MCC950 and Cyclosporin A (CsA) were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). JNK inhibitor VIII was purchased from Calbiochem (San Diego, CA). All the chemical stocks were prepared according to SDS information provided by company and stored in − 20 °C. The reagents were dissolved in dimethyl sulfoxide (DMSO) with the final concentration of DMSO was < 0.1%. No effect of DMSO was observed. A specific antibody against S198 phosphorylated NLRP3 was kindly provided by Professor Tao Li from National Center of Biomedical Analysis, China.