Journal: The Journal of biological chemistry

Article Title: Human p300 protein is a coactivator for the transcription factor MyoD.

doi: 10.1074/jbc.271.15.9009

Figure Lengend Snippet: FIG. 2. p300 associates MyoD in vivo. Immunoblot with an anti- MyoD antibody (Santa Cruz) following immunoprecipitation of whole cell extracts prepared from C2C12 myoblast cells in lysis buffer con- taining either 250 mM salt (lanes 2 and 3) or 150 mM salt (lanes 1, 4, and 5) with an anti-p300 polyclonal antiserum (5 ml). NRbS, controls for immunoprecipitation with normal rabbit serum. WCL, whole cell ex- tract without immunoprecipitation.

Article Snippet: The membrane was blocked with 5% non-fat dry milk in TBS-T buffer (containing 20 mM Tris-Cl, pH 7.6, 137 mMNaCl, and 0.5% Tween 20) for 1 h, incubated with anti-MyoD antibodies (Santa Cruz) diluted in TBS-Twith 3% the drymilk for 1 h, washed in TBS-T, further incubated for 1 h with a secondary antibody conjugated to horseradish peroxidase in TBS-T, and finally washed in TBS-T.

Techniques: In Vivo, Western Blot, Immunoprecipitation, Lysis

Journal: Frontiers in Pharmacology

Article Title: Miya Improves Osteoarthritis Characteristics via the Gut-Muscle-Joint Axis According to Multi-Omics Analyses

doi: 10.3389/fphar.2022.816891

Figure Lengend Snippet: Sequences of all primers.

Article Snippet: The total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 1 h. After washing three times with 1 × buffer composed of 1 ml Tween-20 in 1 L 1× phosphate-buffered saline, the membranes were incubated with anti-AMPK antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States), anti-Chrna1 antibody (1:1,000, Proteintech, Rosemont, IL, United States), anti-Ldh antibody (1:1,000, Abcam, Cambridge, United Kingdom), anti-Mcad antibody (1:1,000, Proteintech), anti-Myod antibody (1:1,000, Proteintech), anti-Tfam antibody (1:1,000, Proteintech), and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight.

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Miya Improves Osteoarthritis Characteristics via the Gut-Muscle-Joint Axis According to Multi-Omics Analyses

doi: 10.3389/fphar.2022.816891

Figure Lengend Snippet: Effects of MY on the expressions of related genes in the tibia muscle of different groups. mRNA expression of AMPK (A) , Ldh (B) , Lcad (C) , Mcad (D) , Tfam (E) , Myod (F) , Murf1 (G) , Chrna1 (H) , Chrnd (I) , Rapsyn (J) , Agrin (K) , and IL-1β (L) . * p < 0.05, compared with the control group; # p < 0.05, compared with the OA group.

Article Snippet: The total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 1 h. After washing three times with 1 × buffer composed of 1 ml Tween-20 in 1 L 1× phosphate-buffered saline, the membranes were incubated with anti-AMPK antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States), anti-Chrna1 antibody (1:1,000, Proteintech, Rosemont, IL, United States), anti-Ldh antibody (1:1,000, Abcam, Cambridge, United Kingdom), anti-Mcad antibody (1:1,000, Proteintech), anti-Myod antibody (1:1,000, Proteintech), anti-Tfam antibody (1:1,000, Proteintech), and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight.

Techniques: Expressing, Control

Journal: Frontiers in Pharmacology

Article Title: Miya Improves Osteoarthritis Characteristics via the Gut-Muscle-Joint Axis According to Multi-Omics Analyses

doi: 10.3389/fphar.2022.816891

Figure Lengend Snippet: Effects of MY on the expressions of related proteins in the tibia muscle of different groups. (A) Protein bands visualized by western blotting. Protein expression of AMPK (B) , Myod (C) , Tfam (D) , Chrna1 (E) , Ldh (F) , and Mcad (G) . * p < 0.05, compared with the control group; # p < 0.05, compared with the OA group.

Article Snippet: The total protein samples (20 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and blocked with 5% skim milk at 37°C for 1 h. After washing three times with 1 × buffer composed of 1 ml Tween-20 in 1 L 1× phosphate-buffered saline, the membranes were incubated with anti-AMPK antibody (1:1,000, Cell Signaling Technology, Danvers, MA, United States), anti-Chrna1 antibody (1:1,000, Proteintech, Rosemont, IL, United States), anti-Ldh antibody (1:1,000, Abcam, Cambridge, United Kingdom), anti-Mcad antibody (1:1,000, Proteintech), anti-Myod antibody (1:1,000, Proteintech), anti-Tfam antibody (1:1,000, Proteintech), and anti-GAPDH antibody (1:10,000, Proteintech) at 4°C overnight.

Techniques: Western Blot, Expressing, Control

Journal: Cells

Article Title: IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

doi: 10.3390/cells12091284

Figure Lengend Snippet: Figure 1. IL-4 treatment promoted myoblast differentiation and increased the expression of myogenic regulatory factors and myomerger in C2C12 cells. (A) Representative immunofluorescence images of MyHC (red) in the differentiated C2C12 cells 72 h after IL-4 application. The nuclei of C2C12 cells were counterstained with DAPI (blue). Scale bars: 50 µm. (B,C) The number of myonuclei per myotube and the differentiation index of IL-4-treated C2C12 cells. N = 3 per group. (D) Representative images of EdU (green) and counterstained nuclei (Hoechst 33342; blue) after 24 h IL-4 treatment in GM. (E) The number of EdU-positive cells significantly decreased in the IL-4-treated C2C12 cells. N = 4 per group. (F) Fold changes in the mRNA levels of MyoD, myogenin, myomaker, and myomerger in the IL-4-treated C2C12 myoblasts. Myoblasts were maintained in DM with 10 ng/mL IL-4. After 72 h incubation, the cells were harvested for qRT-PCR. N = 5 per group. Data are presented as mean ± SD. * p < 0.05.

Article Snippet: Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Expressing, Incubation, Quantitative RT-PCR

Journal: Cells

Article Title: IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

doi: 10.3390/cells12091284

Figure Lengend Snippet: Figure 3. Reduction of IL-4/IL-4Rα signaling suppressed the increased expression of MyoD, myo- genin, and myomerger, but did not affect myomaker expression. (A) Representative immunofluores- cence images of myogenin (red) in the IL-4RαKD C2C12 myoblasts. Cell nuclei were counterstained with DAPI (blue). C2C12 cells transfected with control siRNA (Ctrl) or IL-4Rα siRNA (IL-4RαKD) were grown in GM for 24 h and maintained in DM with or without recombinant IL-4 (10 ng/mL) for 72 h. Scale bars: 50 µm. (B) The percentage of myogenin-positive cells was significantly sup- pressed by IL-4Rα knockdown. N = 5 per group. (C–E) Reduction of IL-4/IL-4Rα signaling by IL-4Rα knockdown significantly suppressed the increased expression of MyoD, myogenin, and my- omerger, whereas the expression of myomaker was not affected. Cells were treated as described in (A). (C,E) show fold changes in mRNA and protein levels, respectively. N = 6 per group. Representative western blot analysis is shown in (D). Data represent mean ± SD. * p < 0.05.

Article Snippet: Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Expressing, Transfection, Control, Recombinant, Knockdown, Western Blot

Journal: Cells

Article Title: IL-4 Signaling Promotes Myoblast Differentiation and Fusion by Enhancing the Expression of MyoD, Myogenin, and Myomerger.

doi: 10.3390/cells12091284

Figure Lengend Snippet: Figure 4. Stimulation of IL-4/IL-4Rα signaling increased the expression of myogenic regulatory factors and myomerger, but not myomaker, even in GM. (A) Representative immunofluorescence images of myogenin (red) in the IL-4RαKD C2C12 myoblasts. The nuclei were counterstained with DAPI (blue). C2C12 cells were treated as in Figure 3 except that cells were maintained in GM, instead of DM, for 24 h. Scale bars: 50 µm. (B) The percentage of myogenin-positive cells significantly increased by IL-4 treatment, which was suppressed by IL-4Rα knockdown. N = 4 per group. (C) Fold change in the mRNA levels of MyoD, myogenin, myomerger, and myomaker. N = 6 per group. Data represent as mean ± SD. * p < 0.05.

Article Snippet: Primary antibodies used were against phosphorylated MyoD (1:1000; Santa Cruz Biotechnology Inc.), myogenin (1:1000; DSHB), myomaker (1:1000, Abcam, Cambridge, UK), myomerger (1:1000; R&D Systems), IL-4Rα (1:1000; Santa Cruz Biotechnology Inc.), MyHC (1:200; DSHB), and GAPDH (1:2000; Cell Signaling Technology Inc., Danvers, MA, USA).

Techniques: Expressing, Knockdown