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: PLOS One

Article Title: Identification of Galectin-9 (Gal-9) as a B7-H4 binding partner and characterization of their glycosylation-dependent interaction that modulates T cell signaling within a multi-ligand/receptor network

doi: 10.1371/journal.pone.0355964

Figure Lengend Snippet: (A) SPR analysis of Gal-9 binding kinetics to PD-1, TIM-3, CD28, and B7-H4. Human PD-1, TIM-3, and CD28 were fused to the same mouse IgG2a Fc tags. 1.2 µg/mL Gal-9 was injected over a Protein A/G-coated Biacore chip to assess binding kinetics to each immobilized Fc-fusion protein. (B) Flow cytometry analysis of the binding between the four proteins (shown in Panel A) and 293T cells transiently overexpressing either WT Gal-9 or the Gal-9 R65A mutant. ( C ) Cell-surface binding of CD28 to Gal-9 analyzed by Co-IP-Western blotting. Cell-surface expression of CD28 on Jurkat T cells was examined by flow cytometry (left panel); CD28 expression was detected using an anti-CD28 antibody (red), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel); 400 µL of whole cell lysates (WCL) from Jurkat T cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-CD28 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-CD28 antibody from that used for IP, together with an anti-Gal-9 antibody, as indicated. ( D ) Cell-surface binding of B7-H4 to Gal-9 was analyzed by Co-IP-Western blotting. Cell-surface expression of B7-H4 on 293T cells transiently transfected with B7-H4 (293T-B7-H4) was examined by flow cytometry (left panel); B7-H4 expression was detected with an anti-B7-H4 antibody (blue), with a cell-only control included (black). For the Co-IP-Western blotting analysis (right panel), 400 µL of WCL from 293T-B7-H4 cells were incubated with or without 4 µg/mL Gal-9 and 200 mM lactose, followed by IP using an anti-B7-H4 antibody. Bound proteins were eluted and analyzed by Western blotting using a different anti-B7-H4 antibody from that used for IP, and an anti-Gal-9 antibody, as indicated. Anti-GAPDH antibody was used as an internal loading control for Western blotting of WCL samples in (C) and (D). (E) Gal-9-mediated stimulation of pCD28 signaling activity in Jurkat T cells. Jurkat T cells were treated with or without plate-bound B7.1, and Gal-9’s dose-dependent effects on pCD28 were analyzed by western blotting. Fold change (FC) in pCD28 band intensity relative to the CD28 loading control was quantified using ImageJ and GraphPad Prism.

Article Snippet: Western blotting was then performed as described above using an anti-CD28 rabbit monoclonal antibody (ABclonal, A20346), different from that used for immunoprecipitation, and an anti-Gal-9 mouse monoclonal antibody (R&D Systems, MAB20455) as primary antibodies, followed by appropriate HRP-conjugated secondary antibodies: HRP-anti-Rabbit IgG antibody (Jackson ImmunoResearch, 111-035-003), HRP-anti-mouse IgG antibody (Jackson ImmunoResearch, 115-035-003), and HRP-anti-GAPDH antibody (ABclonal, AC035).

Techniques: Binding Assay, Injection, Flow Cytometry, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Expressing, Control, Incubation, Transfection, Activity Assay

Journal: iScience

Article Title: Pentachlorophenol enhances bladder cancer cell invasion through altered protein stability

doi: 10.1016/j.isci.2026.117357

Figure Lengend Snippet:

Article Snippet: Rabbit anti-GAPDH , ABclonal , Cat# A19056; RRID: AB_2863474.

Techniques: Recombinant, CCK-8 Assay, Bicinchoninic Acid Protein Assay, shRNA, Plasmid Preparation, Over Expression, Software, Transfection, Western Blot