Journal: Journal of molecular biology

Article Title: Structural and functional diversity of novel coronin 1C (CRN2) isoforms in muscle.

doi: 10.1016/j.jmb.2009.07.079

Figure Lengend Snippet: Fig. 3. CRN2i3, the 60-kDa slower migrating isoform of CRN2, specifically appears during myogenesis. (a) Time course of differentiation of C2F3 myoblasts. Cells were harvested at the indicated time points (day after initiation of differentiation) and analyzed by immuno- blotting using mAb K6–444 and mAb 203–217 specific for annexin A7 as a control of myogenic differentiation. (b) Myogenic conversion of NIH3T3 fibroblasts using a retroviral vector encoding human MyoD. CRN2 from hMyoD-transduced and untransduced NIH3T3 and hMyoD-transduced, differentiated, and undifferentiated C2F3 myoblasts is shown. Cells were harvested 4 days after transduction or after 5 days of differentiation and analyzed by SDS-PAGE followed by Western blotting using the CRN2 and annexin-A7-specific antibodies. (c) ChIP experiments employing lysates of differentiating C2C12 myoblasts and a monoclonal MyoD antibody confirmed the functional relevance of the highly con- served MyoD binding site identified in exon 1a of the CRN2 gene. The PCR products shown were generated by a primer pair very closely positioned to exon 1a within the CRN2 promoter. Input, cross-linked and digested ge- nomic DNA; H2O, negative control; αIgG, negative control; αMyoD, experiment.

Article Snippet: For ChIP assays, the ChIP-IT Express Enzymatic Kit (Active Motif #53009) was used in conjunction with the ChIP-IT Control Kit Mouse (Active Motif #53011) and the monoclonal antibody against MyoD (NovusBiologicals #NB100-56511).

Techniques: Control, Retroviral, Plasmid Preparation, Transduction, SDS Page, Western Blot, Functional Assay, Binding Assay, Generated, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 2. MyoD is degraded in an ATP-dependent manner by HeLa nucleoplasm. MyoD (400 ng) was added to the reaction mix- tures, and degradation was assayed by Western blotting with anti- MyoD as described under “Experimental Procedures.” The reaction mixtures contained an ATP-regenerating (1ATP) or ATP-depleting (to, 2ATP) system and were incubated for 2 h at 37 °C except for the to reaction, which was kept at 4 °C during the incubation period. The results of five independent assays were quantified. Asterisk (*) indicates significance (, 0.001).

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: Western Blot, Incubation

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 3. Degradation of MyoD in HeLa nucleoplasm is dependent on the 26 S proteasome. Degradation reac- tions were carried out in the presence of either 5 mM ATPgS or 20 mM MG132. The ATPgS was added alone or in addition to the ATP-regenerating system (ATP reg). MG132 dissolved in Me2SO was added to reactions containing the ATP-regenerat- ing system. Me2SO alone was run as a control.

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: Control

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 4. Degradation of myoD in HeLa nucleoplasm is dependent on ubiquitin. Degradation reactions were carried out in the presence of the polyu- biquitin chain terminators MeUb and ubiquitin K48R. Each inhibitor was added in increasing amounts (5, 10, and 15 mg/12.5-ml reaction) to reactions con- taining the ATP-regenerating system (ATP reg). Wild-type ubiquitin (20 mg) was added in 2-fold excess over MeUb or Ub K48R (10 mg) in additional reactions. Both the to and (ATP2) reactions con- tained the ATP-depleting system. The to reactions were kept at 4 °C during the 2-h incubation. Reticulocyte lysate or HeLa nucleoplasm served as the cellular fraction.

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: Ubiquitin Proteomics, Incubation

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 5. HeLa nucleoplasm supports the generation of myoD- ubiquitin conjugates. 35S-Labeled myoD was prepared in a coupled transcription-translation system as described under “Experimental Procedures.” The reaction was thereafter inactivated with NEM/DTT. Conjugation assays were performed in the absence or presence of HeLa nucleoplasm (NE), and in the absence or presence of the proteasome inhibitors ATPgS plus ubiquitin aldehyde (ATPgS), as described in the text. Lanes 1 and 4 were incubated at 4 °C. High molecular mass myoD-ubiquitin conjugates resolved on SDS-PAGE are noted (conj). MyoD is indicated by the arrowhead.

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: Ubiquitin Proteomics, Labeling, Conjugation Assay, Incubation, SDS Page

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 6. High molecular mass ubiquitin conjugates accompany HeLa nucleoplasm-mediated myoD degradation. Degradation re- actions containing 100 ng of myoD and supported by either reticulocyte lysate (left) or nucleoplasm (right) were carried out in the presence of ATP and/or MG132, plus ubiquitin aldehyde (Ubal) and ATPgS as described under “Experimental Procedures.” Each reaction mix was analyzed via SDS-PAGE and immunoblot with antibody to myoD. High molecular mass myoD-ubiquitin conjugates are noted (conj.). MyoD is noted by the arrowhead. Note that in the left lane (1ATP) the myoD band is weak as it is degraded efficiently.

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: Ubiquitin Proteomics, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 7. Immunofluorescent localization of MyoD. HeLa cells were transfected with pClneo MyoD. 24 h later cells were incubated with MG132 or LMB for 2 h as described above. Thereafter, localization of MyoD was determined by indirect immunofluorescence with a poly- clonal anti-MyoD antibody (Santa Cruz) and the resulting images were photographed (magnification, 340) using an Olympus BX60 micro- scope. A 5 control, B 5 MG132 (20 mM), C 5 LMB (10 mM).

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: Transfection, Incubation, Immunofluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: The Nuclear Ubiquitin-Proteasome System Degrades MyoD

doi: 10.1074/jbc.m009388200

Figure Lengend Snippet: FIG. 8. MyoD is degraded in vivo in the presence of leptomycin B. A, HeLa cells were transiently transfected with MyoD and 24 h later were incubated with cycloheximide (100 mg/ml) for the indicated times. The relative amount of MyoD at each time point was determined by 10% SDS-PAGE, followed by Western blotting with anti-MyoD as described under “Experimental Procedures.” The reactions were carried out in the presence of MG132 (20 mM) or leptomycin B (10 nM) as indicated. B, the relative amounts of MyoD were quantified from three independent experiments and the half-lives determined as described under “Experimental Procedures.”

Article Snippet: Immunoprecipitations were performed by incubation with a polyclonal anti-MyoD antibody (1:200 dilution, Santa Cruz, sc-760) followed by incubation with protein ASepharose.

Techniques: In Vivo, Transfection, Incubation, SDS Page, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

doi: 10.1074/jbc.m204291200

Figure Lengend Snippet: FIG. 3. Myostatin-induced quiescent cells are different than reserve cells. A, Western blot analyses of C2C12 myoblasts cultured in differentiation media (DM; containing 2% FBS) with () or without () 5 g/ml myostatin for 96 h. C2C12 myoblasts cultured in DM without myostatin have also been “limited trypsinized” to selectively obtain reserve cells (RC) or myotubes (MT). Total proteins (15 g) extracted from the myoblasts were resolved by 4–12% SDS-PAGE, transferred to nitrocellulose filters, and probed with polyclonal anti-p130, polyclonal anti-Myf5, and polyclonal anti-MyoD antibodies. Filters have also been probed with polyclonal anti-myogenin and monoclonal anti-MHC (MHC) antibodies as markers of differentiation as well as monoclonal antitubulin antibodies to demonstrate equal loading. B, Northern blot analysis of C2C12 myoblasts cultured as described above. Total RNA (12 g) extracted from the myoblasts was fractionated by 1% agarose-RNA gel electrophoresis and transferred to Hybond N membrane and probed with 32P-labeled cDNA probes for p130, myf5, and myoD. Ethidium bromide-stained 18 S and 28 S rRNA bands from the gel have been included to demonstrate equal loading and RNA integrity.

Article Snippet: The following primary antibodies were used for immunoblotting: p130, 1:500 dilution of purified rabbit polyclonal anti-p130 antibody (sc317); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (sc304; Santa Cruz Biotechnology Inc.) or 1:150 dilution of purified mouse monoclonal anti-MyoD antibody (554130; Pharmingen); Myf5, 1:200 dilution of purified rabbit polyclonal anti-Myf5 antibody (sc302; Santa Cruz Biotechnology Inc.); myogenin, 1:200 dilution of purified rabbit polyclonal anti-myogenin antibody (sc576; Santa Cruz Biotechnology Inc.); MHC, 1:2000 dilution of purified mouse monoclonal anti-MHC antibody (MF20; gift of Donald Fischman); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; Pharmingen); p-Smad 2/3, 1:150 dilution of purified rabbit polyclonal anti-p-Smad 2/3 antibody (sc11769; Pharmingen); Smad 3-FLAG, 1:500 dilution of purified mouse monoclonal anti-FLAG M2 antibody (F-3165; Sigma); -tubulin, 1:3000 dilution of purified mouse monoclonal anti- -tubulin antibody (DM 1A; Sigma).

Techniques: Western Blot, Cell Culture, SDS Page, Northern Blot, Nucleic Acid Electrophoresis, Membrane, Labeling, Staining

Journal: Journal of Biological Chemistry

Article Title: Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

doi: 10.1074/jbc.m204291200

Figure Lengend Snippet: FIG. 4. MyoD-MRFs and muscle differentiation markers are inhibited by myostatin. A, Western blot analyses of C2C12 myoblasts cultured in growth media (GM; containing 10% FBS) or differentiation media (DM; containing 2% FBS) with () or without () 5 g/ml myostatin for 96 h. Total proteins (15 g) extracted from the myoblasts were resolved by 4–12% SDS-PAGE, transferred to nitrocellulose filters, and probed with polyclonal anti-MyoD, polyclonal anti-Myf5, polyclonal anti-myogenin, monoclonal anti-p21, and monoclonal anti-MHC (MHC) antibodies. Filters have also been probed with monoclonal antitubulin antibodies to demonstrate equal loading. B, Northern blot analysis of C2C12 myoblasts cultured as described above. Total RNA (12 g) extracted from the myoblasts was fractionated by 1% agarose RNA gel electrophoresis and transferred to Hybond N membrane and probed with 32P-labeled cDNA probes for myoD, myf5, and p21. Ethidium bromide-stained 18 S and 28 S rRNA bands from the gel have been included to demonstrate equal loading and RNA integrity.

Article Snippet: The following primary antibodies were used for immunoblotting: p130, 1:500 dilution of purified rabbit polyclonal anti-p130 antibody (sc317); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (sc304; Santa Cruz Biotechnology Inc.) or 1:150 dilution of purified mouse monoclonal anti-MyoD antibody (554130; Pharmingen); Myf5, 1:200 dilution of purified rabbit polyclonal anti-Myf5 antibody (sc302; Santa Cruz Biotechnology Inc.); myogenin, 1:200 dilution of purified rabbit polyclonal anti-myogenin antibody (sc576; Santa Cruz Biotechnology Inc.); MHC, 1:2000 dilution of purified mouse monoclonal anti-MHC antibody (MF20; gift of Donald Fischman); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; Pharmingen); p-Smad 2/3, 1:150 dilution of purified rabbit polyclonal anti-p-Smad 2/3 antibody (sc11769; Pharmingen); Smad 3-FLAG, 1:500 dilution of purified mouse monoclonal anti-FLAG M2 antibody (F-3165; Sigma); -tubulin, 1:3000 dilution of purified mouse monoclonal anti- -tubulin antibody (DM 1A; Sigma).

Techniques: Western Blot, Cell Culture, SDS Page, Northern Blot, Nucleic Acid Electrophoresis, Membrane, Labeling, Staining

Journal: Journal of Biological Chemistry

Article Title: Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

doi: 10.1074/jbc.m204291200

Figure Lengend Snippet: FIG. 5. Myostatin can inhibit MyoD- MRFs and muscle differentiation markers after the myogenic differen- tiation program has been initiated. A, Western blot analyses of C2C12 myoblasts cultured in differentiation media (DM; containing 2% HS) for 48 h, with myosta- tin (8 g/ml) added to the media 0, 6, 12, and 24 h after the switch to DM. C2C12 myoblasts cultured in DM for 0, 6, 12, 24 and 48 h without myostatin were also an- alyzed in the Western blots. Total pro- teins (15 g) extracted from the myo- blasts were resolved by 4–12% SDS- PAGE, transferred to nitrocellulose filters, and probed with polyclonal anti- MyoD, polyclonal anti-Myf5, polyclonal anti-myogenin, monoclonal anti-p21, and monoclonal anti-MHC (MHC) antibodies. Filters have also been probed with mono- clonal anti-tubulin antibodies to demon- strate equal loading. B, Northern blot analysis of C2C12 myoblasts cultured as described above. Total RNA (12 g) ex- tracted from the myoblasts was fraction- ated by 1% agarose-RNA gel electrophore- sis and transferred to Hybond N

Article Snippet: The following primary antibodies were used for immunoblotting: p130, 1:500 dilution of purified rabbit polyclonal anti-p130 antibody (sc317); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (sc304; Santa Cruz Biotechnology Inc.) or 1:150 dilution of purified mouse monoclonal anti-MyoD antibody (554130; Pharmingen); Myf5, 1:200 dilution of purified rabbit polyclonal anti-Myf5 antibody (sc302; Santa Cruz Biotechnology Inc.); myogenin, 1:200 dilution of purified rabbit polyclonal anti-myogenin antibody (sc576; Santa Cruz Biotechnology Inc.); MHC, 1:2000 dilution of purified mouse monoclonal anti-MHC antibody (MF20; gift of Donald Fischman); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; Pharmingen); p-Smad 2/3, 1:150 dilution of purified rabbit polyclonal anti-p-Smad 2/3 antibody (sc11769; Pharmingen); Smad 3-FLAG, 1:500 dilution of purified mouse monoclonal anti-FLAG M2 antibody (F-3165; Sigma); -tubulin, 1:3000 dilution of purified mouse monoclonal anti- -tubulin antibody (DM 1A; Sigma).

Techniques: Western Blot, Cell Culture, SDS Page, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

doi: 10.1074/jbc.m204291200

Figure Lengend Snippet: FIG. 7. The forced expression of MyoD in C2C12 myoblasts can- not rescue myostatin-inhibited myogenic differentiation. West- ern blot analyses of C2C12 myoblasts were transiently transfected with pcDNA3 alone (vector) or pcDNA3-MyoD expression vector (MyoD) and cultured in differentiation media (DM; containing 2% HS) with () or without () 8 g/ml myostatin for 72 h. Total proteins (15 g) extracted from the myoblasts were resolved by 4–12% SDS-PAGE, transferred to nitrocellulose filters, and probed with polyclonal anti-MyoD, mono- clonal anti-MyoD, polyclonal anti-myogenin, monoclonal anti-p21, and monoclonal anti-MHC antibodies. Filters have also been probed with monoclonal antitubulin antibodies to demonstrate equal loading.

Article Snippet: The following primary antibodies were used for immunoblotting: p130, 1:500 dilution of purified rabbit polyclonal anti-p130 antibody (sc317); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (sc304; Santa Cruz Biotechnology Inc.) or 1:150 dilution of purified mouse monoclonal anti-MyoD antibody (554130; Pharmingen); Myf5, 1:200 dilution of purified rabbit polyclonal anti-Myf5 antibody (sc302; Santa Cruz Biotechnology Inc.); myogenin, 1:200 dilution of purified rabbit polyclonal anti-myogenin antibody (sc576; Santa Cruz Biotechnology Inc.); MHC, 1:2000 dilution of purified mouse monoclonal anti-MHC antibody (MF20; gift of Donald Fischman); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; Pharmingen); p-Smad 2/3, 1:150 dilution of purified rabbit polyclonal anti-p-Smad 2/3 antibody (sc11769; Pharmingen); Smad 3-FLAG, 1:500 dilution of purified mouse monoclonal anti-FLAG M2 antibody (F-3165; Sigma); -tubulin, 1:3000 dilution of purified mouse monoclonal anti- -tubulin antibody (DM 1A; Sigma).

Techniques: Expressing, Transfection, Plasmid Preparation, Cell Culture, SDS Page

Journal: Journal of Biological Chemistry

Article Title: Myostatin Inhibits Myoblast Differentiation by Down-regulating MyoD Expression

doi: 10.1074/jbc.m204291200

Figure Lengend Snippet: FIG. 8. The down-regulation of MyoD is mediated through Smad 3. A, West- ern blot analyses of C2C12 myoblasts cul- tured in differentiation media (DM; con- taining 2% HS) for 9 h, with () or without () myostatin (8 g/ml) added to the media after 6 h. Myoblasts cultured in differenti- ation media for the 6 h prior to treatment are referred to as 0 h. Total proteins (15 g) extracted from the myoblasts were re- solved by 4–12% SDS-PAGE, transferred to nitrocellulose filters, and probed with polyclonal anti-p-Smad 2/3 antibodies. Fil- ters have also been probed with mono- clonal antitubulin antibodies to demon- strate equal loading. B, Western blot analyses of Smad 3-FLAG co-immunopre- cipitated with MyoD-V5 from transfected C2C12 myoblasts cultured in differentiation media (DM; containing 2% HS) for 24 h and treated with () or without () myostatin (8 g/ml) for 6 or 12 h. Protein was immu- noprecipitated with monoclonal anti-V5 antibody, resolved by 4–12% SDS-PAGE, transferred to nitrocellulose filters, and probed with monoclonal anti-FLAG anti- bodies. Western blot analyses also confirm comparable levels of Smad 3-FLAG and MyoD-V5 in the total protein extracts used in the immunoprecipitation studies. C, i, C2C12 myoblasts, co-transfected with p3TP-Lux and dominant-negative Smad 3 (dn Smad 3) or empty vector (control vec- tor) were cultured in differentiation media (DM; containing 2% HS) with () or with- out (f) 2 g/ml TGF-1 for 24 h. ii, C2C12 myoblasts, co-transfected with the pJH17- myoD promoter-reporter construct and dominant-negative Smad 2 (dn Smad 2), dominant-negative Smad 3 (dn Smad 3), or empty vector (control vector) were cultured in differentiation media (DM; containing 2% HS) with () or without (f) 2 g/ml myostatin for 24 h. Luciferase activity was determined and normalized to -galacto- sidase activity from the transient transfec- tion of the -galactosidase vector pCH110. The values, denoted as the percentage of the activity in nonmyostatin-treated cells, are the average of triplicate luciferase ac- tivities from two independent experiments. Error bars correspond to the S.E. mean.

Article Snippet: The following primary antibodies were used for immunoblotting: p130, 1:500 dilution of purified rabbit polyclonal anti-p130 antibody (sc317); MyoD, 1:200 dilution of purified rabbit polyclonal anti-MyoD antibody (sc304; Santa Cruz Biotechnology Inc.) or 1:150 dilution of purified mouse monoclonal anti-MyoD antibody (554130; Pharmingen); Myf5, 1:200 dilution of purified rabbit polyclonal anti-Myf5 antibody (sc302; Santa Cruz Biotechnology Inc.); myogenin, 1:200 dilution of purified rabbit polyclonal anti-myogenin antibody (sc576; Santa Cruz Biotechnology Inc.); MHC, 1:2000 dilution of purified mouse monoclonal anti-MHC antibody (MF20; gift of Donald Fischman); p21, 1:400 dilution of purified mouse monoclonal anti-p21 antibody (SX118; Pharmingen); p-Smad 2/3, 1:150 dilution of purified rabbit polyclonal anti-p-Smad 2/3 antibody (sc11769; Pharmingen); Smad 3-FLAG, 1:500 dilution of purified mouse monoclonal anti-FLAG M2 antibody (F-3165; Sigma); -tubulin, 1:3000 dilution of purified mouse monoclonal anti- -tubulin antibody (DM 1A; Sigma).

Techniques: Cell Culture, SDS Page, Western Blot, Transfection, Immunoprecipitation, Dominant Negative Mutation, Plasmid Preparation, Control, Construct, Luciferase, Activity Assay

Journal: Journal of Nanobiotechnology

Article Title: Conductive MXene/adECM hydrogel promotes skeletal muscle regeneration and innervation through Ca 2+ influx modulation and neuromuscular junction formation

doi: 10.1186/s12951-026-04077-y

Figure Lengend Snippet: Biocompatibility and myogenesis-promoting effects of MXene/adECM hydrogels in vitro. ( a ) CCK-8 assay of L6 cells cultured in the conditioned mediums (CMs) at 24 h and 48 h ( n = 3). AM100, AM300, and AM500 respectively refer to 100 µg mL − 1 , 300 µg mL − 1 , and 500 µg mL − 1 MXene/adECM hydrogel. ns : not significant, * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001, compared with Control group, while # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, compared between hydrogel groups. ( b ) Live/dead staining of L6 cells cultured in adECM and MXene/adECM hydrogels on day 1, 3, and 7. Scale bar: 100 μm. ( c ) Immunofluorescence staining of L6 cells cultured on glass substrate and in hydrogels after 10 days of myogenic differentiation induction. MHC (red) indicates the myogenic differentiation. DAPI (blue) stains nuclei. Scale bar: 100 μm. ( d ) Western blotting evaluation of myogenic regulatory proteins (Desmin, Myod1, Myf5) expressed by L6 cells in 2D culture or 3D culture within adECM and MXene/adECM hydrogels. ( e ) Quantitative analysis of western blotting ( n ≥ 3). ns : not significant, * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001, compared with 2D group, while # p < 0.05, ## p < 0.01, compared between hydrogel groups

Article Snippet: After blocking with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4 °C with primary antibodies against Desmin (1:100000; ab32362, Abcam, UK), Myod1 (1:1000; 18943-1-AP, Proteintech, China), and Myf5 (1:10000; ab125078, Abcam, UK).

Techniques: In Vitro, CCK-8 Assay, Cell Culture, Control, Staining, Immunofluorescence, Cell Characterization, Western Blot