Rabbit anti pcdc2 tyr15

Cells were harvested, washed with PBS, and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.1% SDS, 1% NP-40, and 1% Triton X-100) supplemented with 1 mM PMSF (Sigma) and a protease inhibitor cocktail (Roche) at 4°C for 20 min. The lysates were then centrifuged for 15 min at 12,000 rpm at 4°C. The supernatants were collected, and an equal volume of 2X Laemmli’s buffer was added. The sample was boiled for 5 min at 95°C. Proteins were resolved by 10 or 12.5% SDS-PAGE and then transferred to nitrocellulose membranes (Pall Corporation). Membranes were blocked with 5% non-fat milk in TBST (0.1% Tween 20) for 1 h before incubation with primary and secondary antibodies sequentially. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. The following antibodies were used: rabbit anti-FOP (Abcam, ab156013, 1:2,000), mouse anti-GFP (Santa Cruz, sc-9996, 1:5,000), rabbit anti-AURKA (Cell signaling Technology, 14475, 1:2,000), rabbit anti-Cyclin A2 (Abcam, ab18159, 1:10,000), rabbit anti-pCDC2 (Tyr15) (Cell Signaling Technology, 9111, 1:2,000), rabbit anti-pRb (Ser807/811) (Cell Signaling Technology, 8516, 1:2,000), and mouse anti-β-actin (Sigma, A5441, 1:5,000).

Cell culture medium (Dulbecco's modified minimum essential medium, DMEM), fetal bovine serum (FBS), penicillin G, streptomycin, glutamine, sodium pyruvate and Hoechst 33342 were from GIBCO Invitrogen (Carlsbad, CA); rabbit anti p-cdc2(Tyr15) was from Cell Signaling (Danvers, MA), mouse anti cdc2p34 and cyclin D1 were from Santa Cruz (Dallas, TX); Matrigel and Transwell inserts were from BD (Franklin Lakes, NJ); Encapsome and Clodrosome were from Encapsula NanoSciences (Nashville, TN); 3-(4, 5-Dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide (MTT) salt, DMSO, peanut oil, SKA-31, pLKO.1 lentiviral shRNA clones targeting human and murine KCa3.1 mRNA and all other chemicals were from Sigma-Aldrich (St. Louis, MO) or Pierce (Rockford, IL). TRAM-34 was synthesized in our lab as previously described [3 (link)]. NSC95397 was from Enzo Life Sciences, Inc. (New York, NY).