Mouse anti cyclin d3

Tissues and cells were lysed in the RIPA buffer supplemented with a phosphatase inhibitor cocktail (Applygen Technologies, China) and PMSF. A total of 20 µg protein extracts were subjected on the 12% SDS-polyacrylamide gel for electrophoresis, and then transferred onto the polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk for 1 hour at room temperature and incubated with the following primary antibodies at 4°C overnight: rabbit anti-Kisspeptin antibody(KISS1) (Abcam, USA), rabbit anti-progesterone receptor (PR) (Santa Cruz, USA), mouse anti-cyclin D3 (Cell Signaling, USA), and rabbit anti-β-actin(Santa Cruz, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized via enhanced chemiluminescence (Pierce, USA). The relative band intensity was acquired by using the Quantity One software. The data were corrected for background, normalized to β-actin expression.

Sources were: YM155 (Selleck Chemicals); rabbit anti-Survivin IgG (#AF886) (R&D Systems); rabbit anti-P-Rb (S807/811, #9308), rabbit anti-P-Cyclin D1 (T286, #3300), mouse anti-Cyclin D1 (#2926), rabbit anti-Mcl-1(#5453), mouse anti-Cyclin D3 (#2936), rabbit anti-P-Akt (T308, #6955), rabbit anti-Akt (S473, #9271), rabbit anti-rS6 (#2217), rabbit anti-P-rS6 (S235/236, #2211), rabbit anti-P-p70S6K1 (T389, #9205), rabbit anti-mTOR (#2972), rabbit anti-P-mTOR (S2448, #2971), rabbit anti-Raptor (#4978, #4972) rabbit anti-P-Raptor (S792, #2083) rabbit anti-Rictor (#2140), rabbit anti-P-Rictor (T1135, #3806), rabbit anti-AMPKα (#5831), rabbit anti-P-AMPKα (T172, #2535) rabbit anti-AMPKα1 (#2795), rabbit anti-AMPKα2 (#2757), rabbit anti-4E-BP1 (#9644), rabbit anti-P-4E-BP1 (S65) (Cell Signaling Technologies); rabbit anti-Rb (sc-50), rabbit anti-Cyclin D1 (sc-753), rabbit anti-Cyclin D2 (sc-593), mouse anti-GAPDH (sc-51907), mouse anti-α-tubulin (sc-5386), mouse anti-P-rS6 (S240, sc-293143), mouse anti-rS6 (sc-74459), mouse anti-p70S6Kα (sc-39367) (Santa Cruz Biotechnologies, Inc.); mouse anti-β-actin (#A-5441), cycloheximide (CHX), actinomycin D (Act D), MG132, curcumin, (Sigma-Aldrich, Inc.); calyculin A, rapamycin and Compound C (LC labs, Inc.) DMEM/F12 (Media Tech); characterized fetal bovine serum (FBS) (HyClone).