Journal: Journal of Sport and Health Science

Article Title: Exercise attenuates stress-related signaling as sensed by higher phosphorylation of small heat shock proteins in skeletal muscle from older individuals

doi: 10.1016/j.jshs.2025.101111

Figure Lengend Snippet: Relative HSP abundances in whole skeletal muscle homogenates from young adults and older adults pre and post HIT exercise. Representative Westen blots of (A) HSP72, HSP27, and αB-crystallin and (B) phosphorylated HSP27 Ser15 (pHSP27 Ser15) and pαB-crystallin Ser59 in whole muscle homogenates from the vastus lateralis of the same individuals. Calibration curves of mixed muscle homogenates are indicated and were used to determine the relative number of given proteins (see Methods). Stain-free gels are indicative of total protein loading, and molecular weights are indicated by markers collected under white light capture without moving the membrane between that and chemiluminescence detection. Relative abundances of (C) HSP72, (D) HSP27, (E) pHSP27 Ser15, (F) αB-crystallin, and (G) pαB-crystallin Ser59 from young (circle) and older adults Pre (square) and older adults Post (triangle) HIT exercise are shown relative to average Old (pre) on a given gel (data are presented as mean ± SD). Individuals indicated by the number of symbols ( n : 5–7), with the same color assigned to the same individual and consistent across all graphs. * p ≤ 0.05 indicates Brown-Forsye and Welch’s and post hoc analysis using Games-Horwell. HIT = high-intensity training; HSP = heat shock protein; pαB-crystallin Ser59 = phospho-αB-crystallin at Serine59; pHSP27 Ser15 = phospho-HSP27 at Serine15.

Article Snippet: Details of antibodies used are as follows: HSP72 (1 in 500 mouse monoclonal, SMC100A; StressMarq Biosciences, Victoria, Canada); HSP27 (1 in 1000 mouse monoclonal, G3.1 ab2790; Abcam, Cambridge, UK); pHSP27 Ser15 (1 in 2000 monoclonal rabbit, ab76313; Abcam), pHSP27 Ser82 (1 in 2000 polyclonal mouse, ADI-SPA-524; Enzo Biochem, Farmingdale, NY, USA), αB-crystallin (1 in 1000 mouse monoclonal, SPA-222; StressGen Biotechnologies), pαB-crystallin Ser59 (1 in 1000 rabbit polyclonal, SPA-227; StressGen Biotechnologies).

Techniques: Staining, Membrane

Journal: Journal of Sport and Health Science

Article Title: Exercise attenuates stress-related signaling as sensed by higher phosphorylation of small heat shock proteins in skeletal muscle from older individuals

doi: 10.1016/j.jshs.2025.101111

Figure Lengend Snippet: HSP abundances in type I and II skeletal muscle fibers from young and older adults. (A, C, and F) The MHC isoform present was determined in individual muscle fiber segments from the vastus lateralis and, following pooling into type I and type II groups from a given biopsy, were analyzed by Westen blotting. Westen blots of (A) HSP72, (C) HSP27 and pHSP27 Ser15, (F) αB-crystallin and pαB-crystallin Ser59, with MHC isoforms in groups of fibers. Stain-free gels are indicative of total protein loading, and molecular weights are indicated by markers collected under white light capture without moving the membrane between that and chemiluminescence detection. Calibration curves of mixed muscle homogenates are indicated. Relative protein abundances of (B) HSP72, (D) HSP27, (E) pHSP27 Ser15, (G) αB-crystallin, and (H) pαB-crystallin Ser59 in fibers from young (circle) and older adults (square) type I fibers (no outline) and type II fibers (outline). All fibers are expressed relative to the average older adult’s type I fibers. The same color is assigned to the same individual and is consistent with (data are presented as mean ± SD). * p < 0.05 and ** p < 0.01, mixed effect model Univariant using either Tukey’s or Games-Horwell’s multiple comparison test (see Methods). HIT = high-intensity training; HSP = heat shock protein; MHC = myosin heavy chain; pαB-crystallin Ser59 = phospho-αB-crystallin at Serine59; pHSP27 Ser15 = phospho-HSP27 at Serine15.

Article Snippet: Details of antibodies used are as follows: HSP72 (1 in 500 mouse monoclonal, SMC100A; StressMarq Biosciences, Victoria, Canada); HSP27 (1 in 1000 mouse monoclonal, G3.1 ab2790; Abcam, Cambridge, UK); pHSP27 Ser15 (1 in 2000 monoclonal rabbit, ab76313; Abcam), pHSP27 Ser82 (1 in 2000 polyclonal mouse, ADI-SPA-524; Enzo Biochem, Farmingdale, NY, USA), αB-crystallin (1 in 1000 mouse monoclonal, SPA-222; StressGen Biotechnologies), pαB-crystallin Ser59 (1 in 1000 rabbit polyclonal, SPA-227; StressGen Biotechnologies).

Techniques: Staining, Membrane, Comparison

Journal: Journal of Sport and Health Science

Article Title: Exercise attenuates stress-related signaling as sensed by higher phosphorylation of small heat shock proteins in skeletal muscle from older individuals

doi: 10.1016/j.jshs.2025.101111

Figure Lengend Snippet: HSP abundances in type I and II skeletal fibers from older adults pre- and post HIT exercise. Relative protein abundances of (A and B) HSP72, (C and D) HSP27, (E and F) pHSP27 Ser15, (G and H) αB-crystallin, and (I and J) pαB-crystallin Ser59 in fibers from old pre and old post HIT exercise. All fibers are expressed relative to the average old pre type I fibers or relative pre type II depending on fiber type. The same color is assigned to the same Individual, consistent in both graphs and all figures. * p < 0.05 and ** p < 0.01 indicated significant difference in paired t -test (except pHSP27 Ser15 Wilcoxon match-pair rank test). Representative blots are shown in . HIT = high-intensity training; HSP = heat shock protein; pαB-crystallin Ser59 = phospho-αB-crystallin at Serine59; pHSP27 Ser15 = phospho-HSP27 at Serine15.

Article Snippet: Details of antibodies used are as follows: HSP72 (1 in 500 mouse monoclonal, SMC100A; StressMarq Biosciences, Victoria, Canada); HSP27 (1 in 1000 mouse monoclonal, G3.1 ab2790; Abcam, Cambridge, UK); pHSP27 Ser15 (1 in 2000 monoclonal rabbit, ab76313; Abcam), pHSP27 Ser82 (1 in 2000 polyclonal mouse, ADI-SPA-524; Enzo Biochem, Farmingdale, NY, USA), αB-crystallin (1 in 1000 mouse monoclonal, SPA-222; StressGen Biotechnologies), pαB-crystallin Ser59 (1 in 1000 rabbit polyclonal, SPA-227; StressGen Biotechnologies).

Techniques:

Journal: European Journal of Histochemistry : EJH

Article Title: Ketogenic diet regulates Uch-L1(C) to improve cerebral energy metabolism and cognitive function in Alzheimer's disease mice

doi: 10.4081/ejh.2026.4548

Figure Lengend Snippet: Detection of oxidative stress in the hippocampus and cortex of mice brain. A,B ) Expression of 8-OHdG and 4-HNE in hippocampus and temporal cortex in different groups examined by immunofluorescence images (n=3). C,D ) Analysis of fluorescence intensity of 8-OHdG in hippocampus and temporal cortex respectively. E,F ) Analysis of fluorescence intensity of 4HNE in hippocampus and temporal cortex, respectively. n=3, * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: 4-HNE , Stressmarq , SMC-511D , 1:200 (IF) 1:1000 (WB).

Techniques: Expressing, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) HEK-293T or SH-SY5Y cells expressing TAP-tagged WT VPS35 were subjected to TAP methodology and Western blot analysis. Inputs and TAP fractions were probed with anti-VPS35 antibody. TAP purifies VPS35 more efficiently in HEK-293T cells compared to SH-SY5Y cells. ( B ) HEK-293T cells expressing TAP-tagged WT VPS35 or empty vector (EV) were subjected to TAP methodology followed by SDS-PAGE and silver staining. Endogenous VPS35 is observed slightly below VPS35-TAP band at ∼100 kDa. ( C ) STRING diagram of interacting proteins identified by LC-MS/MS analysis of WT VPS35-TAP. ( D ) HEK-293T cells expressing TAP-tagged WT VPS35 or EV were subjected to TAP methodology with HEPES-based buffers followed by Western blot analysis. Input and VPS35-TAP fractions were probed with anti-VPS35 or anti-VPS26 antibodies to confirm recovery of the retromer and then subjected to LC-MS/MS. ( E ) STRING diagram of interacting proteins identified by LC-MS/MS analysis of WT VPS35-TAP. Outside of the core retromer subunits, no known interacting proteins of VPS35 were identified. ( F ) Proportional Venn diagram demonstrating proteins identified in WT VPS35 TAP experiments using Tris– vs. HEPES-based buffers. ( G ) STRING diagram demonstrating the 7 proteins commonly identified between the two TAP experiments.

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Plasmid Preparation, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) HEK-293T cells expressing TAP-tagged WT VPS35 treated with increasing concentrations of DSP were subjected to affinity purification with either streptavidin or calmodulin resin followed by Western blot analysis under reducing or non-reducing conditions. Input and purified fractions were probed with anti-VPS35 or anti-actin antibodies. Only 0.3 mM DSP treatment preserved binding of TAP-tagged VPS35 to both streptavidin and calmodulin resins. (B) HEK-293T cells expressing TAP-tagged VPS35 treated with 0.3 mM DSP were subjected to TAP methodology followed by Western blot analysis under reducing or non-reducing conditions. Input and TAP fractions were probed with anti-VPS35 or anti-WASH1 antibodies. WT VPS35-TAP eluate was subjected to LC-MS/MS analysis. ( C ) Proportional Venn diagram showing the common interacting proteins of WT VPS35 identified in Tris-based alone vs HEPES-based with DSP TAP experiments. ( D ) Proportional Venn diagram demonstrating proteins identified in HEPES-based WT VPS35-TAP assays with or without DSP treatment. The addition of reversible cross-linking greatly increased the number of interacting proteins. ( E , F ) GO and KEGG pathway analysis using the functional annotation tool DAVID of VPS35-interacting proteins identified using DSP-treated TAP assays in HEK-293T cells overexpressing TAP-tagged VPS35-WT. Top 10 GO terms for each category and top 10 KEGG Pathway terms, based on Bonferroni-corrected p-value and fold enrichment, are displayed. Bubble plots generated using SRplot. ( G ) Functional enrichment analysis using the Cytoscape plug-in ClueGo with GO biological process terms of proteins found in DSP-treated WT VPS35 TAP assays. Pathways with a p-value of <0.05 and a Kappa score of 0.4 were mapped. Two-sided hypergeometric statistical analysis test with Bonferroni step-down p-value correction was used. Node size corresponds to p-value.

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Expressing, Affinity Purification, Western Blot, Purification, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Functional Assay, Generated

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) Soluble hemi-brain extracts from 3-4 month-old WT, heterozygous and homozygous KI mice were subjected to IP with anti-VPS35 antibody (or isotype-matched control anti-V5 IgG) followed by Western blot analysis. Input and IP fractions were probed with anti-VPS35 and anti-VPS26 antibodies, prior to subjecting VPS35 IP samples to LC-MS/MS analysis. ( B ) Correlation analysis of VPS35-interacting proteins from WT vs homozygous D620N VPS35 KI reveal a high degree of correlation (R = 0.995). ( C ) Proportional Venn diagram demonstrating the strong overlap between interacting proteins identified in WT and homozygous D620N VPS35 KI brains. ( D ) Table highlighting peptide intensities across conditions for core retromer subunits, and three interacting proteins depleted in D620N VPS35 KI brain. ( E ) Soluble striatal extracts from 3-4 month-old WT and KI mice were subjected to IP with anti-VPS35 antibody (or isotype-matched control anti-V5 IgG) followed by Western blot analysis. Input and IP fractions were probed with anti-VPS35 and anti-VPS26 antibodies, prior to subjecting VPS35 IP samples to LC-MS/MS analysis. ( F ) Table outlining peptide intensities across conditions for core retromer subunits in striatal tissue of D620N VPS35 KI mice. ( G ) Triton-soluble fractions from striatum of adult WT or D620N VPS35 KI mice were subjected to Western blot analysis to monitor steady-state levels of core retromer subunits (VPS35, VPS26, VPS29), Rab7 and TBC1D5. ( H ) Graphs indicate densitometric analysis of protein levels normalized to actin or β-tubulin and expressed as fold-change compared to WT mice (mean ± SEM, n = 3 mice/group). Data are not significant ( P >0.05) by unpaired, two-tailed Student’s t -test.

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test

Journal: bioRxiv

Article Title: Parkinson’s disease-linked D620N mutation selectively alters the brain-specific protein interactome of VPS35

doi: 10.64898/2026.04.09.717005

Figure Lengend Snippet: ( A ) Soluble striatal extracts from 3-4 month-old WT and D620N VPS35 KI mice were subjected to IP with anti-VPS35 antibody (or isotype-matched control anti-V5 IgG) followed by Western blot analysis. Input and IP fractions were probed with anti-VPS35 and anti-TBC1D5 antibodies confirming a TBC1D5 binding deficit in D620N VPS35 KI brain. Arrows indicate the position of TBC1D5 in the IP VPS35 samples and two non-specific bands in the control IP V5 sample. ( B ) HEK-293T cells expressing V5-tagged VPS35 variants (WT or D620N) were subjected to IP with anti-V5 antibody followed by Western blot analysis. Inputs and IPs were probed with anti-TBC1D5 or anti-V5 antibodies. ( C ) Graph indicates densitometric analysis of TBC1D5 levels in V5 IP, normalized to endogenous levels of TBC1D5 in the input, and expressed as a percent of the WT VPS35 IP condition (mean ± SEM, n = 3-4 experiments). ( D ) Representative confocal immunofluorescent images of HEK-293T cells co-labeled for endogenous TBC1D5 (red) and V5-tagged VPS35 variants (green). ( E ) Graph indicates Pearson’s correlation coefficients for TBC1D5 with WT or D620N VPS35 (mean ± SEM, n = 50 cells/condition). Data were analyzed by unpaired, two-tailed Student’s t -test (* P <0.05).

Article Snippet: The following primary antibodies were used: mouse anti-VPS35 (ab57632, Abcam), mouse anti-VPS35 (SMC-602, stressmarq), mouse anti-V5 (Thermo Fisher), mouse anti-V5-HRP (R96125, Invitrogen), mouse anti-GFP (clone 7.1 and 13.1, Roche), rabbit anti-VPS26 (ab23892, Abcam), goat anti-VPS29 (ab10160, Abcam), rabbit anti-WASH1 (SAB4200372, Sigma-Aldrich), rabbit anti-Rab7 (ab137029, Abcam), mouse anti-actin (MAB1501, Millipore), mouse anti-β-tubulin (Sigma, T5201), rabbit anti-TBC1D5 (ab203896, Abcam) and mouse anti-TBC1D5 (sc-376296, Santa Cruz Biotechnology).

Techniques: Control, Western Blot, Binding Assay, Expressing, Labeling, Two Tailed Test