Cells and muscle tissue were lysed in RIPA buffer (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. Protein lysates were heated at 95°C for 5 min in 5 × SDS sample buffer, then separated by 10% SDS polyacrylamide gel electrophoresis (30 μg each lane), followed by transfer to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, United States) using a Mini Trans-Blot Cell system (Bio-Rad). The membrane was blocked with 5% non-fat milk for 3 h. The primary antibodies were incubated overnight at 4°C. The membranes were washed and incubated with secondary antibodies for 1 h at 37°C followed by visualization by enhanced chemiluminescence (Bio-Rad). Primary antibodies specific for EZH2 (ab3748, 1:1,000; Abcam, Cambridge, United Kingdom), MyoD (sc-760, 1:1,000; Santa Cruz Biotechnology, Dallas, TX, United States), MyoG (sc-12732, 1:200; Santa Cruz Biotechnology), MyHC (sc-376157, 1:3,00; Santa Cruz Biotechnology), Ki67 (ab16667, 1:1,000; Abcam), p21 (sc-6246, 1:1,000; Santa Cruz Biotechnology), and β-actin (sc-4777, 1:1,000; Santa Cruz Biotechnology), along with goat anti-mouse IgG-HRP (sc-2005, 1:3,000; Santa Cruz Biotechnology) and goat anti-rabbit IgG-HRP (sc-2004, 1:3,000; Santa Cruz Biotechnology) secondary antibodies were used to detect protein expression.
Sc 376157
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-376157 is a laboratory equipment product offered by Santa Cruz Biotechnology. The core function of this product is to provide a tool for researchers and scientists to conduct their experiments and studies. No further details about the intended use or specific features of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab2722, by Abcam (2 mentions)
Ab2796, by Abcam (2 mentions)
Ab6002, by Abcam (2 mentions)
Ab83832, by Abcam (2 mentions)
Sab4501982, by Merck Group (2 mentions)
Sc 20641, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab3748, by Abcam (1 mentions)
Enhanced chemiluminescence, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab16667, by Abcam (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Mini trans blot cell system, by Bio-Rad (1 mentions)
Sc 12732, by Santa Cruz Biotechnology (1 mentions)
Sc 4777, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Sc 81648, by Santa Cruz Biotechnology (1 mentions)
Bs 0947r, by Bioss Antibodies (1 mentions)
Fv1200, by Olympus (1 mentions)
6 protocols using sc 376157
1
Western Blot Analysis of Muscle Proteins
2
Immunofluorescence Staining of Stem Cells and Adrenergic Receptors
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Immunofluorescence staining was conducted to detect markers of SCs and ADR. Slides were deparaffinized in xylene, rehydrated, and blocked with bovine serum albumin for 2 h at room temperature. Cells on confocal dishes were fixed with 4% paraformaldehyde, and the confocal dishes or slides were incubated with 0.1% Triton X-100 and primary antibodies at a dilution of 1:50–1:200 for approximately 16 h at 4 °C. The primary antibodies were as follows: PAX7 (sc-81648, Santa Cruz, USA; DF7915, Affinity, USA), MyoD (AF7733, Affinity, USA), Myf5 (39801, Active Motif, USA), TH (511027, Zen Bio, China), NPY (ab112473, Abcam, UK), β2-ADR (bs-0947R, Bioss, China), and MYH (sc-376157, Santa Cruz, USA). The slides were incubated with secondary antibodies (KGAB010, KGAB011, KGAB013, KGAB014, Keygen, China) at room temperature for 2 h at a 1:400 dilution, sealed with DAPI and observed by a confocal scanning microscope (Olympus, FV1200, Japan) or a fluorescence microscope (Konigsallee 9–21, Carl Zeiss Microscopy GmbH, Germany). The fluorescence intensity was quantified using FV10-ASW 4.2 Viewer or ImageJ software.
3
Quantifying Myofiber Morphology on Scaffolds
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Cell morphology was analyzed with cells seeded on the scaffolds (microstructured and flat) and control (commercial plastic plate) (n = 3) at a density of 2.5 × 105 cells/cm2 using standard fluorescence techniques in an inverted microscope (Nikon, Eclipse TS2FL, Japan), staining the cells with rhodamine-phalloidin (1:200; R415; Invitrogen, Thermo Fisher Scientific, USA) and Hoechst 33,342 (1:10,000; H1399; Invitrogen, Thermo Fisher Scientific, USA)32 (link). Myofiber identification was performed using the method proposed by Acevedo et al.15 (link), by immunofluorescence stain of anti-myosin heavy chain (1:500; sc-376157; Santa Cruz Biotechnology, Inc). Myofiber diameter and distribution were analyzed using ImageJ.
4
Quantitative Protein Analysis of Muscle Tissue
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Whole cell extracts from isolated muscle tissue or cultured cells were prepared using RIPA buffer (Elpis-Biotech, Korea). Proteins from the whole cell lysates were separated by 15% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were cut into 3 parts by the size; 6–30 kDa for ubiquitin (9 kDa) and HMGB1 (25 kDa), 30–53 kDa for AQP4 (34 kDa) and atrogin1 (42 kDa), 53–170 kDa for α-tubulin (55 kDa) and MYH (120 kDa) to ensure the same experimental conditions instead of membrane duplication. Thus, all the images of α-tubulin in each panel was used as the same images of loading control. Membranes were probed with each antibody; anti-AQP4 (NBP1-87679, Novus Biologicals, Littleton, CO, USA), anti-atrogin 1/MAFbx (F-9) (sc-166806, Santa Cruz Biotechnology, Dallas, TX, USA), anti-ubiquitin (P4D1) (sc-8017, Santa Cruz Biotechnology), anti-HMGB1 (ab79823, Abcam, Cambridge, MA, USA), anti-myosin heavy chain (MYH) (sc-376157, Santa Cruz Biotechnology), and anti-α-tubulin (ab7291, Abcam) antibody. Immunoreactive proteins were visualized using an Amersham ECL kit (GE Healthcare, NJ, USA) and assessed by using the LAS-3000 Image analyzer (Fuji Film, Tokyo, Japan). The protein amounts were assessed by densitometry using Image J software (National Institutes of Health, Bethesda, MD, USA).
5
Protein Expression Analysis in C2C12 Myoblasts
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C2C12 myoblast cells were homogenized on ice in 0.1% Tween-20 homogenization buffer containing protease inhibitors. Nuclear and cytosolic protein were separated and collected using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacture's instruction (78835, Thermo Fisher Scienti c, USA). 20 µg of protein in each well were separated by 7 or 10% SDS-PAGE gel electrophoresis and transferred onto PVDF membrane (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies, including α-tubulin (T9026, 1:5000, Sigma), Histone H3 (ab6002, 1:500, ABCAM), NFATc1 (ab2796, 1:500, Abcam), NFATc2 (ab2722,1:500, Abcam), NFATc3(ab83832,1:500, Abcam), NFATc4 (SAB4501982, 1:1000, Sigma) and MyHC (sc-20641, sc-376157, 1:500, Santa Cruze) overnight at 4 °C, respectively. Thereafter, the blots were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, anti-goat IgG, 1:10000; Santa Cruz) for 90 min. Protein expression was detected by enhanced chemiluminescence method, and the Image J software was used for gray value analysis 19 .
6
Protein Extraction and Western Blotting
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C2C12 myoblastwere homogenized on ice in 0.1% Tween-20 homogenization buffer containing protease inhibitors. Nuclear and cytosolic proteins were separated and collected using NE-PER Nuclear and Cytoplasmic Extraction Reagents according to the manufacture's instruction (78835, Thermo Fisher Scienti c, USA). 20 µg of protein in each well were separated by 7 or 10 % SDS-PAGE gel electrophoresis and transferred onto PVDF membrane (Millipore). After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies, including α-tubulin (T9026, 1:5000, Sigma), Histone H3 (ab6002, 1:500, ABCAM), MyoG (sc-12732, 1:250, Santa Cruze), MEF2C (5030S, 1:500, CST), NFATc1 (ab2796, 1:500, Abcam), NFATc2 (ab2722,1:500, Abcam), NFATc3(ab83832,1:500, Abcam),NFATc4 (SAB4501982, 1:1000, Sigma) and MyHC (sc-20641, sc-376157, 1:500, Santa Cruze) overnight at 4 °C, respectively. Thereafter, the blots were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG, anti-goat IgG, 1:10000; Santa Cruz) for 90 min. Protein expression was detected by enhanced chemiluminescence method, and the Image J software was used for gray value analysis 19 .
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