C2C12 cells and gastrocnemius muscle were lysed with RIPA buffer (Solarbio, Beijing, China); the protein concentrations were measured by a BCA kit (Beyotime, Shanghai, China). Then, 40 µg of the samples were electrophoresed in 10–12% SDS polyacrylamide gels and electrically transferred to 0.2-µm PVDF membranes (Millipore, USA). The membranes were immersed and incubated in 5% fat-free milk at room temperature for 1 hour. The membranes were then incubated with primary antibodies against HDAC2, P53, P21, IKK, and NF-κBp65 (1:1000, Cell Signaling Technology, USA) and MURF1, MAFbx, and SMP30 (1:1000, Abcam, UK) overnight at 4°C, followed by incubation with fluorescent secondary antibodies (1:1000, Cell Signaling Technology, USA). The GAPDH antibody (1:1000, Cell Signaling Technology, USA) was used as the control. Fluorescence signal detection was performed with an infrared imaging instrument (Odyssey, USA), and protein expression was quantified by densitometry analysis with ImageJ software.
Mafbx
Manufactured by Abcam
Sourced in United States, United Kingdom
MAFbx, also known as Atrogin-1, is a protein that functions as an E3 ubiquitin ligase, a key component of the ubiquitin-proteasome system. It plays a role in the regulation of protein degradation and muscle atrophy.
Automatically generated - may contain errors
Lab products found in correlation
Murf1, by Abcam (4 mentions)
Fluorescent secondary antibody, by Cell Signaling Technology (2 mentions)
Ripa buffer, by Solarbio (2 mentions)
Smp30, by Abcam (2 mentions)
Hdac2, by Cell Signaling Technology (2 mentions)
0.2 m pvdf membrane, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Eif4e, by Cell Signaling Technology (1 mentions)
Pras40, by Cell Signaling Technology (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
Murf1, by Santa Cruz Biotechnology (1 mentions)
Bnip3, by Cell Signaling Technology (1 mentions)
Mitofilin, by Abcam (1 mentions)
Chemiluminescence kit, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
6 protocols using mafbx
1
Western Blot Analysis of Muscle Proteins
2
Western Blotting Protein Expression Analysis
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Western blotting was used to verify the expressed proteins as previously described.18 (link) Briefly, gastrocnemius and C2C12 cells were lysed with RIPA buffer (Solarbio, Beijing, China), and protein was quantified by using a BCA protein assay kit (Beyotime, Shanghai, China). Forty micrograms of the samples were separated on 10–12% SDS‐polyacrylamide gels and electrophoretically transferred onto PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk in Tris‐buffered saline (TBS) for 1 hour, the membranes were incubated with primary antibodies against HDAC2, P53, P21, IKK, NF-κBp65 (all 1:1000, Cell Signaling Technology, USA), MURF1, MAFbx, and SMP30 (all 1:1000, Abcam, UK) overnight at 4 °C overnight, followed by incubation with fluorescent secondary antibodies (1:1000, Cell Signaling Technology, USA) for 1 hour. Quantification of signal intensities was quantified using ImageJ software.
3
Immunoblotting of Signaling Proteins
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For immunoblotting, primary antibodies against Akt (Ser473, #9271; total, #9272), PRAS40 (Thr246, #2997; total, #2691), TSC2 (Thr1387, #5584; total, #3635), mTOR (Ser2448, #2971; total, #2983), S6K1 (Thr389, #9234; total #2708), rpS6 (Ser235/236 #2211), 4E-BP1 (Thr37/46, #2855; total, #9644), eIF4e (total, #9742), eEF2 (Thr56, #2331; total, #2332), AMPK (Thr172, #4188; total, #2532) and p38 (Thr180/182, #9211; total, #9212) were all purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against total MAFbx (#92281) and REDD1 (#63059) were purchased from Abcam (Cambridge, UK) and total MuRF-1 (#sc-32920) antibody from Santa Cruz Biotechnology (Heidelberg, Germany). All primary antibodies were diluted 1:1000, except in the case of phospho-eEF2, where the dilution was 1:2000. Secondary anti-rabbit antibodies (#7074; 1:10,000) were purchased from Cell Signaling Technology and secondary anti-goat antibodies (#ab7132; 1:10,000) from Abcam.
4
Immunoblotting for Protein Expression
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For immunoblotting, primary antibodies against androgen receptor (#D6F11XP®), mTOR (#2983), S6K1 (#2708), eEF2 (#2332), AMPK (#2532), ULK1 (#6439), p62 (#8025), LC3B (#2775), Beclin (#3495) and BNIP3 (#44060), were purchased from Cell Signaling Technology (Beverly, USA). Primary antibodies against 5α-reductase 2 (#293232), MuRF-1 (#sc-398608), UBR5 (#sc-515494), TOM20 (sc-136211) and RPS6 (#sc-74459), were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Primary antibodies against MAFbx (#92281), Porin (#154856), OPA1 (#157457), DRP1 (#184247), FIS1 (#156865), MFN2 (#56889) and Mitofilin (#137057), were purchased from Abcam (Cambridge, UK). All antibodies were diluted 1:1000 except for 5α-reductase, MuRF-1, UBR5, S6, Beclin, LC3B, OPA1, MFN2 which were diluted 1:500, and eEF2 and Porin which were diluted 1:2000. Secondary anti-mouse (#7076, 1:10000) and secondary anti-rabbit (#7074, 1:10000) were purchased from Cell Signaling Technology.
5
Western Blot Analysis of Muscle Proteins
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Proteins were extracted from skeletal muscles using RIPA Lysis Buffer (Cwbio, Beijing, China) with an ultrasonic disintegrator, separated by 10% SDS-poly-acrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with the primary antibody of interest at 4°C overnight, followed by incubation with the corresponding horseradish peroxidase (HRP)conjugated secondary antibody at 4°C for 2 hours. Specific protein bands were visualized using Chemiluminescence kit (Millipore, Billerica, MA, USA) and detected with Light-Capture Tanon-5200 (Yuanpinghao Biotechnology Co., Ltd. Beijing, China). The primary antibodies used in the Western blot were βactin, MAFbx, and MuRF1 (Abcam, Cambridge, MA, USA).
6
Protein Characterization in Cell Lysates
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Cells were lysed with NP40 buffer (25 mM HEPES, 100 mM NaCl, 5 mM MgCl 2 , 10% glycerol, 0.2% NP-40, phosphatase inhibitor cocktail-1 and -2 (Sigma) and protease inhibitor cocktail (Roche) for total protein characterization. When nuclear extraction was required, cells were extracted using a CHEMI-CON Nuclear Extraction Kit (Millipore, #2900). Equal amounts of cell lysate were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and detected using an enhanced chemiluminescence system (Pierce Biotechnology). Primary antibodies used were p62 (Abcam, #ab24609), GAPDH (Cell Signaling, #5174), Ki67 (Abcam, #ab15580), Myogenin (BD Pharmingen, #556358), MuRF1 (Abcam, #ab172479), MAFbx (Abcam, #ab168372), Tubulin (Abcam, #ab6160), MyoD (Santa Cruz Biotechnology, #sc760), H3 (Cell Signaling, #9517), MHC (Millipore, #05-833), IMP1 (Cell Signaling, #2852), IMP2 (MBL, #RN008P), NRas (Santa Cruz, #sc31), and cMyc (Cell Signaling, #5605).
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