Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-ethylene diamine tetra acetic acid (EDTA), phosphate-buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Welgene (Daegu, Republic of Korea). Horse serum was purchased from Gibco (Grand Island, NY, USA). Penicillin–streptomycin was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Myosin heavy chain (MyHC), p-Akt, p-mTOR, MAFbx, MuRF1 and HSC70 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). p-p70S6K and p-4E-BP1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Rapamycin was obtained from Calbiochem (St. Louis, MO, USA). Protocatechuic acid (analytical standard), (+)-catechin hydrate (≥98.0%, HPLC), p-coumaric acid (≥98.0%, HPLC), ellagic acid (≥95.0%, HPLC), chlorogenic acid (≥98.0%, titration), caffeic acid (≥98.0%, HPLC), dimethyl sulfoxide (DMSO), ethyl alcohol, formic acid, and DEX were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Fisher Scientific Inc. (Cleveland, OH, USA), and ferulic acid was obtained from Toronto Research Chemicals (TRC, North York, ON, Canada).
Mafbx
Manufactured by Santa Cruz Biotechnology
Sourced in United States
MAFbx is a laboratory equipment product manufactured by Santa Cruz Biotechnology. It functions as an E3 ubiquitin ligase, which plays a role in protein degradation pathways.
Automatically generated - may contain errors
Lab products found in correlation
Murf1, by Santa Cruz Biotechnology (7 mentions)
P mtor, by Santa Cruz Biotechnology (2 mentions)
Myogenin, by Santa Cruz Biotechnology (2 mentions)
Murf1, by Novus Biologicals (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
β tubulin, by Santa Cruz Biotechnology (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
Ellagic acid, by Merck Group (1 mentions)
Protocatechuic acid, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Horse serum, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Ethyl alcohol, by Merck Group (1 mentions)
P p70s6k, by Cell Signaling Technology (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Catechin hydrate, by Merck Group (1 mentions)
11 protocols using mafbx
1
Assessment of Muscle Cell Signaling
2
Myogenic Marker Expression Analysis
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C2C12 cells were differentiated in DM with the test sample and cell lysates were subjected to Western blotting analysis to determine protein expression of myogenic markers and E3 ligases. Harvested cells were suspended in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail) and proteins were loaded onto sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred to polyvinylfluoride membrane in transfer buffer (39 mM glycine, 48 mM Tris base, 0.037% SDS, and 20% methanol) for 2 h. The membrane was then incubated with antibodies specific to MHC (Santa Cruz Biotechnology, Dallas, TX, USA), MyoD (Santa Cruz Biotechnology), Myogenin (Santa Cruz Biotechnology), MAFbx (Santa Cruz Biotechnology), and MuRF1 (Santa Cruz Biotechnology). p38 MAPK activation was analyzed by using antibodies against phospho-p38 (Cell Signaling Technology, Danvers, MA, USA) and p38 MAPK (Cell Signaling Technology). Pan-cadherin (Sigma, St. Louis, MO, USA) was used as a loading control.
3
Immunoblotting Analysis of Muscle-Related Proteins
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Tissue extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling Technology, USA). The resulting preparations (10 to 20 μg) were then fractionated by polyacrylamide-SDS gel electrophoresis and immunoblotted with SIRT-1 (sc-15404), PGC-1β (sc-373771), ATP5B (sc-55597), MuRF1 (sc-32920), MAFbx (sc-33782), tropomyosin (sc-28543), MYH (sc-20641), hypoxanthine phosphoribosyltransferase (HPRT) (sc-20975), NOX2 (gp91-phox) (sc-130543), and β-actin (47778) from Santa Cruz Biotechnology, Inc., USA; with UCP-3 (catalog no. 97000), pAMPKβ1 (Ser182) (catalog no. 4186), and AMPKβ1 (catalog no. 4150) from Cell Signaling Technology, USA; with ATG5 (NBP2-24389) and LC3B (NB100-2220) from Novus Biologicals, USA; and with anti-SQSTM1/p62 (catalog no. ab56416) from Abcam, USA. The primary and secondary antibodies were used at dilutions of 1:1,000 and 1:10,000, respectively. The blots were visualized with SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA), according to the manufacturer’s instructions. The immunoreactive bands were analyzed using ImageJ software, and a densitometry analysis was conducted.
4
Western Blot Protein Analysis Protocol
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Equal values of protein (150 μg/line) were resolved in SDS-PAGE and relocated to nitrocellulose membranes. Subsequently, the membrane was blocked for 2 h in a saline solution [10 mM Tris (pH 7.5), 150 mM NaCl, 0.05% Tween 20] at room temperature with 5% non-fat milk. Membrane washing (three washes, 10 min each) occurred in saline solution. Then, the membrane was incubated membrane with diluted primary antibody (Akt, mTOR, p70S6k, GSK3, and MAFbx) from Santa Cruz Biotechnology (Santa Cruz, CA, United States) in either 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight. After a new membrane washing process, the membrane was incubated with an anti-IgG antibody (1:10,000 dilution) attached to horseradish peroxidase (saline solution with 1% non-fat milk) for 1 h. Posteriorly, the incubation of the membrane was performed using a substrate for peroxidase and a chemiluminescence enhancer (ECL Western Blotting System Kit, GE Health Care, Little Chalfont, Buckinghamshire, United Kingdom) for 1 min and submitted to X-ray film. After the development of the films, the intensity of the band was measured by optical densitometry (Hatanaka et al., 2013 (link)).
5
Comprehensive Immunoblotting Assay Protocols
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For immunoblotting, primary antibodies against S6K1 (Thr389, #9234; total #2708), mTOR (Ser2448, #2971; total, #2983), 4E‐BP1 (Thr37/46, #2855; total, #9644), eEF2 (Thr56, #2331; total, #2332), JNK (Thr183/Tyr185, #4668), SMAD2 (Ser245/250/255, #3104; total, #5339), TAZ (Ser89, #59971), ACC (Ser79, #3661; total, #3676), AMPK (Thr172, #4188; total, #2532), ULK1 (Ser317, #12753; total, #8054), LC3b (#2775), GABARAP (#13733), BNIP3 (#44060) TSC2 (Ser1387, #5584; total, #3635) PRAS40 (Thr246, #13175; total, #2691) and p38 (Thr180/Thr182, #4511; total, #8690), were purchased from CST (Beverly, MA, USA). Primary antibody for total YAP (sc‐101199), total TAZ (sc‐293183), total JNK (sc‐7345), MuRF‐1 (#sc‐398608), MAFbx (#sc‐16806) and UBR5 (#sc‐515494) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Primary antibody for YAP (Ser127, #PA5‐17481) was purchased from Thermo Scientific. Primary antibody for REDD1 (#63059) was purchased from Abcam (Cambridge, UK). Primary antibody for HIF‐1α (#NB100‐134) was purchased from Novus Biologicals (Abingdon, UK).
All primary antibodies were diluted 1:1000 except for the total eEF2 and total 4E‐BP1, which were diluted 1:2000, and total JNK, total YAP, total TAZ, UBR5, MuRF‐1 and MAFbx which were diluted 1:500. Secondary anti‐rabbit (#7074; 1:10 000) and secondary anti‐mouse (#7076; 1:10 000) were purchased from CST.
All primary antibodies were diluted 1:1000 except for the total eEF2 and total 4E‐BP1, which were diluted 1:2000, and total JNK, total YAP, total TAZ, UBR5, MuRF‐1 and MAFbx which were diluted 1:500. Secondary anti‐rabbit (#7074; 1:10 000) and secondary anti‐mouse (#7076; 1:10 000) were purchased from CST.
6
Autophagy Regulation in Angiotensin II Model
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Angiotensin II, chloroquine (CQ), Bafilomycin A1(Baf A1) and 3-methyladenine (3-MA) were purchased from Sigma Aldrich (Sigma Aldrich, USA). Primary antibodies for detecting Sirt3 (rabbit monoclonal) (D22A3) [5490], FoxO1 (rabbit monoclonal) (C29H4) [2880], LC3 (rabbit polyclonal) [2775], LC3 (rabbit polyclonal) [3868], Beclin-1 (rabbit monoclonal) (D40C5) [3495] were purchased from Cell signalling Technology (CST, UK). Primary antibodies against ac-FoxO1 (rabbit polyclonal) (FKHR D19) [sc49437], MuRF1 (mouse monoclonal) [sc398608], MAFBx (mouse monoclonal) (sc166806) were purchased from Santa Cruz Biotechnology (Santa Cruz, USA). Primary antibodies against p62 (mouse monoclonal) [ab56416] was obtained from Abcam. Primary antibodies against β-Tubulin (mouse monoclonal) [BM1453], GAPDH (mouse monoclonal) [BM1623], α-SMA (mouse monoclonal) [BM0002] was purchased from Boster.
7
Western Blot Analysis of Myogenin, MuRF1, and MAFbx
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Proteins (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked at room temperature for 1 h with 1% bovine serum albumin (BSA) in TBS-T (10 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20) and then incubated with primary antibodies against Myogenin, MuRF1, MAFbx and GAPDH (Table I ; all purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at room temperature for 3 h. Membranes were incubated with secondary horseradish peroxidase-conjugated rabbit anti-goat IgG, goat anti-mouse IgG (both Santa Cruz Biotechnology, Inc.) and goat anti-rabbit IgG (Cell Signaling Technology, Inc., Danvers, MA, USA; Table II ) at room temperature for 1 h and bands were detected using an enhanced chemiluminescence western blotting kit (Thermo Fisher Scientific, Inc.). Experiments were performed in triplicate and densitometry analysis was performed using Multi-Gauge software version 3.0 (Fujifilm Life Science, Tokyo, Japan).
8
Antibodies for Protein Signaling Analysis
9
Protein Extraction and Analysis from Tumors
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Proteins from tumors were extracted with a buffer solution (20 mM Tris-HCl, pH 7.5, 2 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 5 μL of a protease inhibitor cocktail (Sigma)). Proteins (20 μg/lane) were separated on a 12.5% polyacrylamide gel (a precast SDS gel (Bio-Rad)) and then transferred to a polyvinylidene difluoride membrane (Immobilon, Millipore). Proteins were determined using antibodies against mouse IL-6 (1:200, Abcam), TNF (1:200, Santa Cruz Biotechnology), Myostatin (1:100, Abcam), MAFbX (1:200, Santa Cruz Biotechnology), MuRF 1 (1:100, Novus), Cathepsina L (1:100, Abcam), Calpain (1:100, Santa Cruz Biotechnology), p-IκB-α (1:100, Santa Cruz Biotechnology) and FOXO1 (1:200, Abcam). The antibodies then were stripped off the membrane and re-probed with a specific antibody against β-actin (1:5000, Novus Biologicals). The intensity was quantified using the Fotodyne Image analysis System (Fotodyne, Hartland, WI, USA) and the TotalLab software (Nonlinear Dynamics, Durham, NC, USA).
10
Comprehensive Immunoblotting Analysis of Muscle Proteins
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Total protein of the cell or tissue samples was harvested using radioimmunoprecipitation lysis buffer after washing three times with PBS. Immunoblotting was carried out according to our previous method (10 (link)). The primary antibodies targeted the following proteins: MyHC (1:500; no. MAB4470; R and D Systems), MyoD (1:500; no. NB100-56511; Novus Biologicals), AKT (1:1000; no. 9272S; CST), p-AKT (Ser473) (1:2000; no. 4060S; CST), PI3K (p55α) (1:1000; no. 11889S; CST), p-PI3K p55 (Tyr199) (1:1000; no. 4228S; CST), β-tubulin (1:200; no. sc-58880; Santa Cruz Biotechnology), MyoG (1:1000; no. NB100-56510SS; Novus Biologicals), MuRF1 (1:200; no. sc-398608; Santa Cruz Biotechnology), MAFbx (1:200; no. sc-166806, Santa Cruz Biotechnology), cyclin E (1:100; no.sc-377100; Santa Cruz Biotechnology), cyclin D1 (1:100; no. sc-450; Santa Cruz Biotechnology), PTEN (1:500; no. sc-7974; Santa Cruz Biotechnology), IGF2BP2 (1:100; no. sc-377014; Santa Cruz Biotechnology), and proliferating cell nuclear antigen (1:100; no. sc-56; Santa Cruz Biotechnology). The secondary antibodies included goat anti-rabbit IgG (1:3000; no. BA1054; BosterBio) and goat antimouse IgG (1:3000; no. BA1050; BosterBio).
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