Journal: Journal of Sport and Health Science

Article Title: Weightlifting outperforms voluntary wheel running for improving adiposity and insulin sensitivity in obese mice

doi: 10.1016/j.jshs.2025.101100

Figure Lengend Snippet: Resistance exercise exceeds the benefits of endurance exercise in ameliorating metabolic dysfunction. Following 8 weeks of diet and exercise interventions, all mice were assessed for their metabolic function by GTT, ITT, and skeletal muscle response of Akt and AS160 phosphorylation to injection of insulin measured by Western blot. (A–C) HOMA-IR taken after an overnight fast for baseline glucose and insulin. (D and E) GTT from 0–120 min and calculated AUC; colored * indicates significant difference from NC-SED. (F and G) ITT from 0–60 min and calculated AUC; colored * indicates significant difference from NC-SED. (H–N) pAkt stimulation, AS160 S318, and AS160 T642 in hindlimb muscles before and after insulin injection and the pre–post ∆ in phosphorylation. (O and P) Total and phosphorylated 4E-BP1. (Q–T) Western results for Raptor, COX4, LC3 II/I, and ubiquitin staining. Representative western blot images inset right. Data presented as mean ± standard error of the mean. Statistical analysis performed by analysis of variance between groups: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NC-SED n : 8–16 (white); HFD-SED n : 8–18 (red); HFD-R EX n : 8–16 (blue); HFD-E EX n : 8–15 (green). 4E-BP1 = Eukaryotic translation initiation factor 4E binding protein; Akt = protein kinase B; AS160 = Akt substrate 160; COX4 = cytochrome c oxidase 4; CS = citrate sythase; E EX = endurance exercise; GAPDH = glyceraldehyde 3 phosphate dehydrogenase; GTT = glucose tolerance test; HFD = high fat diet; HOMA-IR = homeostatic model assessment for insulin resistance; iAUC = integrated area under the curve; ITT = insulin tolerance test; LC3 II/I = microtubule-associated protein light chain 3; NC = normal chow; pAkt = phospho-Akt; R EX = resistance exercise; SED = sedentary; Ub = ubiquitin.

Article Snippet: Primary antibodies used for analysis were from Cell Signaling Technologies (Danvers, MA, USA) and diluted 1:1000 unless otherwise stated as follows: protein kinase B (Akt; 1:500; #4691; Cell Signaling Technologies), phospho-Akt (pAkt) S473 (1:500; #9271; Cell Signaling Technologies), Ubiquitin (#3933; Cell Signaling Technologies), microtubule-associated protein light chain 3 (LC3 II/I; #4018; Cell Signaling Technologies), cytochrome c oxidase subunit 4(COX4; #11967; Cell Signaling Technologies), Akt substrate 160 (AS160 S318; #8619; Cell Signaling Technologies), AS160 T642 (#8881; Cell Signaling Technologies), eukaryotic translation initiation factor 4E binding protein (4E-BP1; #9452; Cell Signaling Technologies), and glyceraldehyde 3 phosphate dehydrogenase (GAPDH; #2118; Cell Signaling Technologies).

Techniques: Phospho-proteomics, Injection, Western Blot, Muscles, Ubiquitin Proteomics, Staining, Binding Assay

Journal: bioRxiv

Article Title: Antitoxin-induced auto-phosphorylation neutralizes the nucleotidyltransferase toxin AbiEii from Streptococcus agalactiae to safeguard global translation

doi: 10.64898/2026.01.19.700257

Figure Lengend Snippet: T65 is important, but not essential, for NTase and phosphorylation activity. ( A ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the presence of either 5 µM AbiEii WT or AbiEii-p. ( B ) In vitro transcription/translation reactions assessing levels of DHFR control protein produced in the absence and presence of 5 µM AbiEii T65A, AbiEii D192A, or AbiEii-p. Reactions were loaded onto 4-20% gradient gels ran at 180 V for 1 h. ( C ) Densitometric analysis of SDS-PAGE bands from ( B ) corresponding to DHFR. ( D ) Dose-response curves of increasing concentrations of either AbiEii WT or AbiEii T65A plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4A and B . ( E ) Logarithmic dose-response curves of either 5 µM AbiEii WT or AbiEii T65A incubated with increasing concentrations of AbiEi plotted against DHFR expression, normalized against DHFR expressed in the absence of toxin (100% expression). Data points were generated following densitometric analysis of bands corresponding to DHFR from Supplementary Figs. S4C and D . Logarithmic curves were generated using non-linear regression in Prism (GraphPad). ( F ) Endpoint viable count antitoxicity assays of E. coli DH5α transformed with either pBAD30 empty vector, pTRB482 (AbiEii WT), or pTRB736 (AbiEii T65A), and either pTA100 empty vector or pTRB481 (AbiEi). Overnight cultures were re-seeded into fresh LB supplemented with Ap, Sp and D-glu and grown to mid-log phase. Samples were serially diluted 10 −2 -10 −9 and spotted onto M9A plates containing Ap and Sp, with or without D-glu, L-ara, and IPTG for repression of toxin expression, induction of toxin expression, and induction of antitoxin expression, respectively. Plates were incubated at 37 °C for 48 h, after which they were imaged and colonies counted to determine CFU/ml (one-way ANOVA, P < 0.05). “T” = toxin induced, “TA” = antitoxin + toxin induced. Assays shown are representative of three independent biological replicates and plotted data represent the mean ± SEM.

Article Snippet: Five μM of purified AbiEii WT or AbiEii-p were added to cell-free transcription/translation protein synthesis reactions (PURExpress ® ; NEB) to quantify changes in DHFR control protein expression.

Techniques: Phospho-proteomics, Activity Assay, In Vitro, Control, Produced, SDS Page, Expressing, Generated, Incubation, Transformation Assay, Plasmid Preparation