Anti cyclin d1 hd 11

Frozen specimens were ground in liquid N₂ and dissolved in Triton X-100 lysis buffer. The cells were also dissolved in the same lysis buffer. The extracts were centrifuged at 13,200 rpm for 20 min at 4 °C. The protein concentration in the supernatant was determined by Bradford assay using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein (~150 μg) were separated on 8 ~ 12 % sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gels, transferred to nitrocellulose membranes using a Semi-Dry Transfer Cell (Bio-Rad), hybridized with primary antibody for 2 h at room temperature (RT), identified with a secondary antibody for 1 h at RT, and exposed to Kodak film. Rabbit polyclonal anti-PIAS3 (H-169), mouse monoclonal anti-p-tyr705-STAT3 (B-7), anti-STAT3 (F-2), anti-ERα (D-12), and anti-cyclin D1 (HD-11) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Chromatin immunoprecipitation (ChIP) analysis was conducted as described [8 (link)] following the protocol provided by Upstate Biotechnology (Charlottesville, VA). Chromatin solutions were precipitated overnight at 4 °C with rotation, using 8 μg of anti-FLAG, 4 μg of anti-Cyclin D1 (HD-11, Santa Cruz Biotechnology, Santa Cruz, CA), H3(Ac-K9) (Millipore, #07-352, Billerica, MA), p300, (Santa Cruz Biotechnology, #sc-585X, Santa Cruz, CA), HDAC3 (Abcam, #ab7030, Cambridge, UK), H3(Dimethyl-K9) (Millipore, #1220, Billerica, MA), SUV39H1 (Millipore, #05-615, Billerica, MA) and HP1α (Millipore, #05-685, Billerica, MA). For negative control, rabbit IgG and mouse IgG were immunoprecipitated. ChIP analysis for each immunoprecipitated protein was conducted on the endogenous murine Top2A promoter (35 cycles of PCR). ChIP-DNA quantitation was conducted in an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA), using Power SYBR Green (AB Biosciences) according to the manufacturer’s guidelines. Equal quantities of ChIP-DNA were used for the real-time PCR quantitation. Ct values were used to calculate the relative fold enrichment (2^-ΔCt, ΔCt = Ct^input - Ct^IgG). A one-way ANOVA followed by Student’s t-test comparison was performed to compare the relative fold enrichment (n = 3).