Anti myogenin

Cells were fixed with 4 % paraformaldehyde for 20 min and then permeabilized with 0.3 % Triton-X for 20 min at room temperature. Cells were washed for 5 min with PBST three times after fixing and permeabilization. Cells were incubated in blocking media (PBS-T, 3 % bovine serum albumin, 5 % normal goat serum, 8 % protein concentrate (vector labs), and 0.2 % Tween-20) for 2 h at room temperature. Cells were incubated with primary antibody solution in blocking media with antibodies overnight at 4 °C at the following dilutions: 1:200 anti-fast MyHC (Sigma M4276) and 1:100 anti-slow MyHC (Sigma M8421), anti-Pax7 (DSHB, 1/15), anti-MyoD (1/20, sc-760, clone M-318), and anti-Myogenin (1/50, BD Biosciences-556358). After thorough washing (three times, 15 min each in PBST), the secondary antibody solution containing 1:250 goat anti-mouse 488 (Lifetech) or 1:250 goat anti-rabbit 488 (Lifetech) and Hoechst 33342 (Thermofisher) in blocking buffer was incubated for 1 h at room temperature (dilutions used for all immunofluorescent antibodies had previously been titrated in C2C12 differentiated myotubes). Images of sorted cells were obtained using an Axio-Observer inverted light microscope and AxioVision software (Zeiss, 2015). A merged multidimensional acquisition was set up using a 365-nm reflector for Hoechst and a 470-nm reflector for MyHC, Pax7, MyoD, and MyoG.

Cells were lysed with RIPA buffer (50 mM Tris pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, 1% SDS, 150 mM NaCl, 2 mM EDTA, 1 × protease inhibitor cocktail, and 1 × phosphatase inhibitor cocktail). Total protein levels were determined using the Bio-Rad protein assay kit, and equal amounts of proteins were loaded and separated on SDS-PAGE. After transferring to PVDF membrane (Millipore), proteins were detected using a standard immune-blotting technique. The following primary antibodies were used: anti-Pax7 (Aviva Systems Biology, ARP32742, 1:400), anti-Myf5 (Santa Cruz, sc-302, 1:200), anti-Myogenin (BD Biosciences, 556358, 1:250), anti-phospho-ERK1/2 (Cell Signaling, 4370, 1:1000), anti-ERK1/2 (Cell Signaling, 9107, 1:1000), anti-phospho-p38 (Cell Signaling, 9216, 1:1000), anti-p38 (Cell Signaling, 9212, 1:1000), anti-integrin α7 (Calbiochem, ST1637, 1:500), anti-gpihbp1 (Thermo Scientific, PA1-16976, 1:250), anti-GAPDH (Abcam, ab9484, 1:1000), and anti-β-actin (Sigma, A5441, 1:2000). Target proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce). The density of target protein bands was quantified using NIH ImageJ software. The expression of phospho-ERK1/2 and phospho-p38 was normalized to ERK1/2 and p38, respectively. The expression of all other proteins was normalized to GAPDH and/or β-actin.

The protocol of western blot has been described before [33] . Briefly, Aliquots of total lysate (100 µg) in RIPA buffer were run on 8% SDS-PAGE gels before blotted onto PVDF membrane. Then, PVDF membranes were extensively washed with 1X PBS containing 0.5% Tween 20 (PBST) before blocked by blocking solution (5% BSA in PBST) for an hour. Both primary and HRP-conjugated secondary antibodies were diluted 1∶1000 in blocking solution and incubated sequentially with the blot. After extensive washes with PBST, the signals was detected by a chemilminescence kit (Amersham Pharmacia Biotech) and visualized on X-ray films (Super RX, Fuji Medical X-film; Tokyo, Japan). For detection of internal control, all the blots were stripped and washed thoroughly in PBST, then, blocked and incubated with Gapdh or Lamin B1 antibody as described above. The extraction of nuclear protein has been described before [34] (link). Polyclonal and monoclonal FoxO1 antibodies were purchased from Cell Signaling Technology (#9462 and # 2880 respectively). Other antibodies used in this study include anti-β-catenin (MAB1329, R&D systems), anti-Mef2 (SC-133, Santa Cruz), anti-MyoD (554130, BD Pharmingen), anti-Myogenin (556358, BD Pharmingen), Lamin B1 (ab16048, ABcam).