C2C12 mouse myoblasts and HEK293 cells were purchased from ATCC (Manassas, VA). High-glucose DMEM, fetal bovine serum and horse serum were from GIBCO (Grand Island, NY). FuGENE 6 Transfection Reagent was from Roche (Indianapolis, IN). Immobilized protein A plus was from PIERCE (Rockford, IL). Long R3 IGF-1 (recombinant analog) and protease inhibitor cocktail for mammalian tissues (P8340) were from Sigma-Aldrich (St. Louis, MO). Wortmannin and rapamycin were from Calbiochem (San Diego, CA). U0126 was from LC Laboratories (Woburn, MA). Protein assay dye reagent concentrated was from Bio-Rad Laboratories (Hercules, CA). Glutathione sepharose 4B, ECL and ECL plus solutions were obtained from GE Healthcare (Piscataway, NJ). Antibodies: phospho-S6K1 (T389), phospho-S6K1 (T421/S424), Akt, phospho-Akt (T308), phospho-Akt (Ser473), IRS, phospho-IRS (S636/S639), PRAS40, phospho-PRAS40 (T246), phospho-GSK3α/β (S21/9), TSC1, phospho-TSC2 (S939), phospho-TSC2 (T1462), phospho-ERK1/2 (T202/T204), ERK1/2 and Rheb were from Cell Signaling Technology (Danvers, MA). GSK3β was from BD Transduction Laboratories (San Jose, CA). Tuberin (C-20) and S6K1 (C-18) were from Santa Cruz Biotechnology (Santa Cruz, CA). 14.3.3, Pan Ab-4 (CG15) was from Thermo Scientific (Fremont, CA). The ANTI-FLAG polyclonal antibody was from Sigma-Aldrich (Saint Louis, MO). Rabbit polyclonal to myc tag and mouse monoclonal (CH-19) to pan-cadherin were from abcam (Cambridge, MA). GST antibody was from Bethyl Laboratories (Montgomery, TX). Peroxidase labeled anti-rabbit IgG and anti-mouse IgG secondary antibodies were from Vector Laboratories (Burlingame, CA). Plasmids: pcDNA3.1-myc-TSC1 (12133), pcDNA3-Flag-TSC2 (14129), pcDNA3-Flag-TSC2-S939A (14132), pcDNA3-Flag-TSC2-T1462A (14130) and pGEX-2TK-14-3-3 beta GST (13276) were purchased from Addgene (Cambridge, MA).
Cell culture and transfection
All cell culture experiments were performed in a humidified environment at 37 °C in a 5% CO2
. The skeletal muscle cell line C2C12 myoblasts were grown in DMEM supplemented with 10% fetal bovine serum and penicilin-streptomicin at low confluence. Myoblasts were transfected while the cells were in suspension [32
]. To induce differentiation, culture medium was switched to 2% horse serum when cells were fully confluence. IGF-1 treatment:
C2C12 myotubes at 4 days of differentiation were treated with serum/antibiotics free media for 120 min and then stimulated for 60 min with recombinant IGF-1 (100 ng/ml in serum/antibiotics free DMEM), co-incubated with/without wortmannin (10 μM), rapamycin (50 nM) or U0126 (10 μM). Series of the experiments were repeated at least 3 times with using different passage of C2C12 myotubes.
Immunoprecipitation assay and western blotting
To study the functional interactions between TSC1, TSC2 and/or 14-3-3, co-immunoprecipitation assays were performed as described previously [33
]. CHAPS based buffer (0.3% CHAPS, 40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM B-glycerophosphate, 50 mM NaF and 10 μl/ml protease inhibitor cocktail) was used to produce total cell lysates. One milligram of total protein was used from cell lysates and samples were immunoprecipitated with each antibody and immobilized protein A. Immunocomplexes were washed for three times with CHAPS based buffer and then washed once with wash buffer (50 mM HEPES (pH 7.5), 40 mM NaCl and 2mM EDTA). Precipitated protein samples were then subjected to SDS-PAGE. Western blotting assay was carried out as previously described [33
The C2C12 myoblasts were plated in 60 mm diameter cell culture dish and then differenetiated to myotubes for 4days. C2C12 myotubes were washed twice with PBS and then scraped off in 3 ml of ice-cold buffer (20 mM Tricine (pH 7.8), 250 mM sucrose, 1 mM EDTA (pH 8.0) and 10 μl/ml protease inhibitor cocktail). The samples were homogenized with using a dounce homogenizer for 20 times, and then centrifuged at 1,000 xg for 10 min at 4 °C. The pellet was collected as nuclei-enriched fraction, and the supernatant was then separated by ultracentrifugation at 300,000 xg for 30 min at 4 °C. After ultracentrifugation, the supernatant (cytosolic fraction) was removed and the pellet (membrane fraction) was diretly lysed in 200 μl of 1× SDS-PAGE sample buffer. The supernatant (cytosolic fraction) protein sample was concentrated by acetone precipitation and then re-suspended in 1× SDS-PAGE sample buffer at 1.0 μg/μl concentration. 10 μl of each fractionated sample was loaded onto the gel.
GST-14-3-3 pull-down assay
For the GST-14-3-3 binding experiments, expression and purification of GST fusion protein was performed according to the manufacturer’s instructions (GST Gene Fusion System Handbook, Amersham Biosciences, Piscataway, NJ). C2C12 myotubes were washed twice with PBS and then lysed in the buffer containing 20 mM Tris (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1 mM PMSF and protease inhibitor cocktail. Cleared myotube protein lysates were mixed with the purified GST fusion protein bound to sepharose beads and then incubated for 3 hours at 4 °C. Sepharose beads-protein complex were washed for four times with lysis buffer and then re-suspended in SDS-PAGE sample buffer.
All results are reported as means ± SE. Two groups comparison was determined by student’s T-test, and multi-group comparisons were performed by one-way analysis of variance followed by Tukey’s post-hoc test. For all comparisons, the level of statistical significance was set at p<0.05.