serum response factor (srf) antibody  (Santa Cruz Biotechnology)

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors

doi: 10.3389/fcell.2021.690452

Figure Lengend Snippet: Effects of mechanical stretch on growth and myosin expression in colon-26 (C26)-treated myotubes. (A) Experiment overview: C2C12 myoblasts are plated on in growth media (GM); after 48 h at ∼85% confluence, differentiation media is added, which corresponds to day 0. At day 5 of differentiation, fully formed contractile myotubes are present, and conditioned media (CM; GM or C26 + GM) is diluted with 50% serum-free DMEM and added to wells for 48 h. Myotubes are either left unstretched (0%) or stretched 5% in the last 24 h of CM incubation. (B) The side view of the stretching device; cells adhere to the type I collagen coated Uniflex membrane, and a rubber gasket is placed around each six-well plate to create a tight seal; vacuum pressure is applied to deform the membrane around the loading post, which creates a uniaxial stretch (image adapted from Flexcell International). (C) Myotube diameter of unstretched (0%) and stretched (5%) myotubes treated with GM control or C26 + GM ( n = 216 myotubes/group). (D) Myosin heavy chain (MyHC)-Fast (0% n = 18–21/group; 5% n = 9/group) and (E) MyHC-Slow ( n = 9–11/group) protein expression measured by Western blotting. (F) MyHC-Fast (type IIA, Myh2) ( n = 6/group), (G) MyHC-Fast (type IIB, Myh4) ( n = 6/group), (H) MyHC-Fast (type IIX, Myh1) ( n = 6/group), (I) MyHC-Slow (type I, Myh7) ( n = 6/group), (J) skeletal α-actin (Acta1) ( n = 6/group), and (K) Myocyte Enhancer Factor 2C (MEF2C) ( n = 6/group) mRNA expression calculated using 2 –ΔΔ Ct method with GAPDH as a housekeeping gene. (L) Representative Western blotting images with Coomassie blue stain as a protein loading control. Data are presented as means ± SEM as a fold change from GM 0% stretch. Two-way ANOVAs were performed to determine differences. & Main effect of stretch; # main effect of C26 + GM; *significant from GM 0% stretch; # significant from GM 5% stretch; ^significant from C26 + GM 0% stretch. Statistical significance set at p ≤ 0.05.

Article Snippet: After blocking, primary antibodies for phosphorylated STAT3 (Y705) 1:1,000, total STAT3 1:2,000, phosphorylated p38 (T180/Y182) 1:1,000, total p38 1:2,000, phosphorylated ERK1/2 (T202/Y204) 1:1,000, total ERK1/2 1:2,000, phosphorylated rpS6 (S240/244) 1:1,000, and total rpS6 1:2,000, phosphorylated Akt (S473) 1:1,000, total Akt 1:2,000, phosphorylated 4E-BP1 (T37/46) 1:1,000, total 4E-BP1 1:2,000, phosphorylated SAPK/JNK (T183/Y185) 1:1,000, total SAPK/JNK 1:2,000 (Cell Signaling Technology, Danvers, MA, United States), Atrogin-1/MAFbx, MuRF-1 1:1,000 (ECM Biosciences, Versailles, KY, United States), and MyHC-Fast (protein stain for fast type II fibers; corresponding genes: MyH1 and MyH2) and MyHC-Slow 1:4,000 (protein stain for slow type I fibers; corresponding gene MyH7), serum response factor (SRF) 1:500, myogenin 1:500, and MyoD 1:500 (Santa Cruz Biotechnology, Dallas, TX, United States), integrin β1D 1:1,000, and puromycin 1:2,000 (Millipore, Billerica, MA, United States) were incubated overnight at 4°C in 5% non-fat milk–TBST.

Techniques: Expressing, Incubation, Membrane, Control, Western Blot, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Effect of Mechanical Stretch on Myotube Growth Suppression by Colon-26 Tumor-Derived Factors

doi: 10.3389/fcell.2021.690452

Figure Lengend Snippet: C2C12 myotube growth suppression by colon-26 (C26) tumor-derived factors on type I collagen-coated silastic membranes. (A) Experiment overview: C2C12 myoblasts are plated on type I collagen-coated silastic Uniflex membranes in growth media (GM). After 48 h at ∼85% confluence, differentiation media (DM) is added, which corresponds to day 0 of differentiation. On day 5 of differentiation, myotubes are fully formed, and conditioned media (CM: GM, C26 + GM) diluted with 50% serum-free DMEM is added to wells for 48 h. Myotubes are imaged Pre and Post 48-h incubation with DM, GM, or C26 + GM for diameter measurements. Cells are harvested for protein and RNA on day 7 of differentiation. (B) Representative × 20 brightfield images. (C) Myotube diameter quantification Pre (day 5; n = 162 myotubes) and Post 48 h DM, GM, or C26 + GM incubation ( n = 162–378 myotubes/group). (D) Myosin heavy chain (MyHC)-Fast (type II fibers) and MyHC-Slow (type I fibers) protein expression measured by Western blotting (Fast: n = 18–21/group; Slow: n = 9/group). (E) MyHC-Fast type IIA (Myh2), type IIB (Myh4), type IIX (Myh1), MyHC-Slow type I (Myh7), and skeletal muscle α-actin (Acta1) mRNA expression ( n = 6/group) calculated using the 2 –ΔΔ Ct method with GAPDH as a housekeeping gene. (F) Ratio of phosphorylated (p-) to total protein expression of signal transducer and activator of transcription 3 (STAT3 Y 705 ) ( n = 12–15/group); p38 T 180/Y182 mitogen-activated protein kinase (MAPK) ( n = 6/group), and extracellular signal-regulated kinase 1/2 (ERK1/2 T 202/Y204 ) ( n = 6/group) measured by Western blotting. (G) Representative Western blotting images: dashed line represents different areas of the same gel. Data are presented as means ± SEM as a fold change from GM. One-way ANOVA (C) or Student’s unpaired t -test was performed to determine differences. Lowercase letters (a, b, and c) denote significant difference between groups; *significant from GM control. Statistical significance set at p ≤ 0.05.

Article Snippet: After blocking, primary antibodies for phosphorylated STAT3 (Y705) 1:1,000, total STAT3 1:2,000, phosphorylated p38 (T180/Y182) 1:1,000, total p38 1:2,000, phosphorylated ERK1/2 (T202/Y204) 1:1,000, total ERK1/2 1:2,000, phosphorylated rpS6 (S240/244) 1:1,000, and total rpS6 1:2,000, phosphorylated Akt (S473) 1:1,000, total Akt 1:2,000, phosphorylated 4E-BP1 (T37/46) 1:1,000, total 4E-BP1 1:2,000, phosphorylated SAPK/JNK (T183/Y185) 1:1,000, total SAPK/JNK 1:2,000 (Cell Signaling Technology, Danvers, MA, United States), Atrogin-1/MAFbx, MuRF-1 1:1,000 (ECM Biosciences, Versailles, KY, United States), and MyHC-Fast (protein stain for fast type II fibers; corresponding genes: MyH1 and MyH2) and MyHC-Slow 1:4,000 (protein stain for slow type I fibers; corresponding gene MyH7), serum response factor (SRF) 1:500, myogenin 1:500, and MyoD 1:500 (Santa Cruz Biotechnology, Dallas, TX, United States), integrin β1D 1:1,000, and puromycin 1:2,000 (Millipore, Billerica, MA, United States) were incubated overnight at 4°C in 5% non-fat milk–TBST.

Techniques: Derivative Assay, Incubation, Expressing, Western Blot, Control