Fig 2: Differentially expressed phosphoproteins (DPPs) (Rps6, Pten, Ywhag, mTOR, Magi1, and Pkn2) participate in the PI3K‐AKT signalling pathway were significantly different in the therapeutic mechanism of hydrogen in CLP‐induced mice. (A) The western blot were used to identify the differentially expressed phosphoproteins and the total proteins. The results showed there were no changes in the total level of Ywhag, mTOR, and Magi1 quantified by the ratio of band density to those of β‐actin, respectively (B) and the MS/MS spectrums from Phosphorylated peptides. (C) (p‐Ywhag assigned to TSADGNEKK; p‐Magi1 assigned to AENEVPSPASSHHSSNQPASLTEEK; p‐mTOR assigned to IMLRMAPDYDHLTLMQKVEVFEHAVNNTAGDDLAK). The b and y ions including loss of ammonia and water were considered when we calculated the PTM score.) (D) The expression of Rps6, p‐Rps6, Pten, p‐Pten, mTOR, p‐mTOR, Pkn2, and p‐Pkn2 in CLP + H2 were detected by western blot. Quantitative analysis of p‐Rps6, p‐Pten, p‐mTOR, and p‐Pkn2 is shown as the ratio of band density to that of Rps6, Pten, mTOR, and Pkn2, respectively. NS, no significance; *p < 0.05; **p < 0.01; ***p < 0.001. Magi1, membrane‐associated guanylate kinase1; mTOR, mammalian target of rapamycin; Pkn2, protein kinase N2; Pten, phosphatase and tensin homologue deleted on chromosome ten; Rps6, Ribosomal protein S6; Ywhag/14–3‐3, tyrosine 3‐Monooxygenase/tryptophan 5‐monooxygenase activation protein gamma

Fig 3: Cytoplasmic NFATc3 facilitates VSMC contractile-to-synthetic phenotype switching by promoting global protein synthesis. (A) Representative images and quantification of NFATc3 and ACTA2 expression using immunofluorescence staining in MASMCs isolated from mice treated with vehicle and BAPN for 28 days (n = 6). (B) NFATc3-associated protein subcellular localization based on LC–MS/MS. (C) Gene Ontology analysis of NFATc3-associated proteins identified using LC–MS/MS. (D) Polysome profiling shows NFATc3 distribution in MASMCs using sucrose density gradient centrifugation (n = 3). Western blot of NFATc3 in the elution profiles. (E) Representative Western blot images and quantification of puromycin incorporation assays from MASMCs isolated from Nfatc3-KIfl/fl and Nfatc3smcKI mice (n = 4). (F) HPG incorporation assay in VSMCs isolated from the aortas of Nfatc3-KIfl/fl and Nfatc3smcKI mice treated with BAPN for 28 days (n = 4). (G) Western blot and quantification of ACTA2, CNN1, SM22α, and OPN in MASMCs treated with PDGF-BB for 24 h before a 12-h CHX treatment (n = 4). (H) Representative Western blot images and quantification of ACTA2, CNN1, SM22α, and OPN in MASMCs isolated from aortas of Nfatc3-KIfl/fl and Nfatc3smcKI mice and treated with PDGF-BB (20 μg/L) for 24 h before a 12-h CHX (50 μmol/L) treatment (n = 4). (I) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. Western blot of RPL28 and RPS6 levels. Data are presented as mean ± SD. (E) Unpaired Student’s t-test; two-tailed P-values. (G) Two-way ANOVA with Tukey’s correction; adjusted P-values. (H) One-way ANOVA with Tukey’s correction; adjusted P-values.

Fig 4: Substrate phosphorylation of PKA and RpS6 content and phosphorylation in muscle following clenbuterol or placeboPKA substrate phosphorylation (A) and protein abundance (B) and phosphorylation (C) of RpS6 in men 2 h after ingestion of clenbuterol (CLEN, n = 11) or placebo (PLA, n = 10) at day 1 and day 14 of treatment. Bars are mean and circles represent data from individual participants.

Fig 5: Substrate phosphorylation of PKA and RpS6 content and phosphorylation in muscle following clenbuterol or placeboPKA substrate phosphorylation (A) and protein abundance (B) and phosphorylation (C) of RpS6 in men 2 h after ingestion of clenbuterol (CLEN, n = 11) or placebo (PLA, n = 10) at day 1 and day 14 of treatment. Bars are mean and circles represent data from individual participants.