Specific primary antibody

The procedure for protein expression analysis was followed by Western blotting technique as described earlier [14 (link)]. Briefly, testis protein from each samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Each membrane was incubated for 1 h in Tris-buffered saline containing 0.1% Tween-20 and 5% skim milk to block nonspecific binding. The membranes were then incubated with specific primary antibodies (1:1000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA). The internal control used was β-Actin. The proteins were detected using horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence detection system (GE Healthcare Life Sciences, Little Chalfont, UK).

Standard procedure was followed for western blotting as described previously [41 (link)] with some modifications. Briefly, tumor lysates were prepared by homogenization in lysis buffer [40 mM HEPES, 50 mM KCl, 1% Triton X-100, 1 mM Na₃VO₄, 50 mM NaF, 5 mM EDTA, 1 mM phenyl-methylsulfonyl fluoride, 1 mM benzamidine 1% Triton-X and complete protease inhibitor cocktail (Sigma)]. 40 μg of protein lysate per sample was loaded and resolved onto 10 or 12% SDS polyacrylamide gel and transferred to PVDF membrane. The levels of PI3K, Akt, phospho-Akt (Ser473), Glut-1, Hypoxia-inducible transcription factor-1α (HIF-1α) and β-actin were determined using specific primary antibodies (Santa Cruz Biotechnology, CA), followed by treatment with the appropriate peroxidase conjugated secondary antibodies. Immunoreactive bands were visualized using chemiluminescent reagent and captured using Microchemi (DNR Bioimaging Systems, Israel) gel documentation system.

E2 and progesterone were purchased from Sigma Co. (USA). A specific shRNA against cyclin G1 (shCyclin G1) was designed and synthesized by GenePharma (Shanghai, China). Scrambled shRNA was also synthesized as a negative control. Specific primary antibodies against cyclin G1 and glyceraldehyde-phosphate dehydrogenase (GAPDH) were commercially purchased from Santa Cruz Biotech. (USA). Breast cancer MCF-7 cells were obtained from American Type Culture Collection (ATCC, USA). Cells were maintained in RPMI 1640 (Gibco, USA) containing 10% fetal bovine serum (Gibco) at 37°C in humidified atmosphere of 5% CO₂.

At the end of treatment (i.e., 60 min) in each group, portions of the gastrocnemius muscles were taken as samples to analyze the insulin signaling proteins, IRS-1 and PPARγ. Muscle samples were homogenized in buffer solution before centrifugation at 21,880 × g. The supernatants were used to estimate the amount of protein using an assay kit from Bio-Rad Laboratories. The supernatant (i.e., protein) was mixed with 4× sodium dodecyl sulfate loading dye and boiled for 15 min at 95°C for denaturation. Separating (8%) and stacking gels were prepared. Protein in buffer (90 μg/mL) was subsequently loaded into each well for electrophoresis. Proteins were electrophoretically transferred to polyvinylidene difluoride membranes at 4°C. The membranes were then blocked with 5% nonfat dry milk in phosphate-buffered saline for 1 h at room temperature and incubated with specific primary antibodies (Santa Cruz Biotechnology, Inc.). After the membranes were washed in buffer containing 0.1% Tween 20 in 1× phosphate-buffered saline, the blots were incubated with horseradish peroxidase-linked specific secondary antibody (Santa Cruz Biotechnology, Inc.) and evaluated using an enhanced chemiluminescence detection using ECL Reagent Plus (PerkinElmer Life Sciences, Inc.). Band intensities were quantified by densitometry to observe the target proteins.

Protein lysates from liver tissues or HepG2 cells were prepared using Pro-Prep Protein Extraction Solution (Intron Biotechnology, Seoul, Korea) according to the manufacturer’s instructions. The protein lysates (50 μg) were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Amersham, UK). The membranes were incubated with specific primary antibodies (1:1000) at 4 °C overnight followed by incubation with anti-rabbit or anti-mouse secondary antibodies (1:1000) (Santa Cruz Biotechnology) conjugated to peroxidase, and protein bands were visualized using an enhanced chemiluminescence system (ECL Advance, GE Healthcare, Hatfield, UK).