Cyclin d1 dcs 6

Preparation of cell lysates, conditions for immunoprecipitation, p21 depletion experiments, histone H1 kinase assays and immunoblotting experiments have been described previously^{17 (link),50 (link)}. To detect P^S317-Chk1, P^S345-Chk1 and phosphorylation-induced mobility shifts of Chk2, pRb and p130, extracts were analyzed on 7.5% SDS-PAGE gels. Other cell cycle regulators and P^T68-Chk2 were analyzed on 12.5% SDS-PAGE gels. For loading controls (LC) the scans of amido-black-stained PVDF membranes for each gel were shown.
Most of the primary antibodies were described in our earlier publications^{17 (link),19 (link),23 (link),51 (link)}. In addition, we have used: cyclin D2 (M-20, Santa Cruz Bio.), cyclin D3 (D-7 and B-10, Santa Cruz Bio), Cdk2 (D-12 and M-2; Santa Cruz Bio.), Cdk4 (C-22, Santa Cruz Bio.), Cdk6 (C-22 and B-10, Santa Cruz Bio.), Cdc6 (180.2, Santa Cruz Bio.), RRM2 (N-18, Santa Cruz Bio.), Cdc25C phospho-S216 (Cell Signalling), cyclin D1 (DCS-6, Cell Signalling), Ki67 (Abcam, ab16667), Rb phospho-S795 (Abcam, ab47474). Secondary antibodies for immunoblot analysis were anti-mouse (DAKO, Glostrup, Denmark) and anti-rabbit IgG HRP-conjugates (Promega, Madison, WI, USA) and protein AG coupled to HRP (Pierce, Rockford, IL, USA). The detection system used was Western Lightning Plus-ECL (PerkinElmer) and Amersham Hyperfilm^TM (GE Healthcare).

Cells were lysed in RIPA buffer [50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) NP40, 0.5% (v/v) Na-deoxycholate, 0.1% (v/v) SDS] with 1 tablet/10 ml Complete Mini EDTA-free protease inhibitors (Roche). SDS-PAGE and immunoblots were performed using standard protocols.
Antibodies against the indicated proteins were used as follows – HIF2α (PA1-16510, Thermo Scientific), Cezanne (custom antibody, Eurogentec), β-actin (3700, Cell Signaling), PHD3 (A300-327A, Bethyl Labs), BNIP3 (ab10433, Abcam), Glut-1 (53519, Anaspec), p52 (05-361, Merck Millipore), E2F1 (3742, Cell Signaling), SP1 (07-645, Upstate-Millipore), cyclin D1 (DCS6, Cell Signaling), cyclin E (HE12, Cell Signaling), cyclin A (C-19, Santa Cruz), GFP (2956, Cell Signaling), cleaved PARP (D214) (9541, Cell Signaling), c-Myc (9E10, Sigma), phosphorylated Chk1 at S345 (2341, Cell Signaling).