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: bioRxiv

Article Title: Rapid Intracellular Delivery of Human Heat Shock Protein 72 Inhibits Neurodegeneration and Oxidative Damage After a Traumatic Brain Injury

doi: 10.64898/2026.05.03.722564

Figure Lengend Snippet: A) Schematic of the CCI method. 100µg of cortical tissue extract from each of the rats were plated in triplicate in the wells of an ELISA plate and probed, as described in Methods, at a 1:2000 dilution with a rabbit monoclonal to the pTau S396 phosphorylation site followed by a goat anti-rabbit conjugated to horseradish peroxidase (GAR-HRP). TMB color development was stopped at 5min for this analysis. B) A scatter plot showing all of the triplicate results from both the ipsilateral (darker color) and contralateral (lighter color) hemispheres of each rat is a daunting task to read. The mean for the three contralateral absorbance values from Rat #35 were 327.15% greater than the mean for the other contralateral results; hence this data set was dropped from our analysis (circled in blue). C) To help visualize the data easier, the same results from each group of rats are presented as box plots with the ipsilateral results on the left and the contralateral on the right. Note that the graphs in (B) and (C) reveal global differences both between treatment groups and within groups (i.e. when comparing ipsi vs. contra). D) It is equally important to analyze biomarker differences between the ipsi- and contralateral hemispheres in each rat individually. This is accomplished by plotting the absorbance ratios against a black line denoting an equal distribution of signal in both hemispheres (ipsi / contra = 1). Analysis of the intra-hemispheric differences for each individual rat reveals a lower pTau S396 neurodegeneration signal level in the ipsilateral for rats treated with Fv-HSP72.

Article Snippet: Rubicon Biotechnology has engineered nearly 20 variants of Fv-HSP72 to improve the therapeutic’s efficacy in a microenvironment that is mildly acidic , and enriched in reactive radicals .

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Biomarker Discovery

Journal: bioRxiv

Article Title: Rapid Intracellular Delivery of Human Heat Shock Protein 72 Inhibits Neurodegeneration and Oxidative Damage After a Traumatic Brain Injury

doi: 10.64898/2026.05.03.722564

Figure Lengend Snippet: Schematics of the 3 Fv-HSP72 variants tested. The ESG and YASYL peptides allow for detection of the 3E10 scFv subunit consisting of a variable light chain (V L ), linker, variable heavy chain (V H ), and a portion of a human constant heavy chain (CH1). These peptides also allow us to differentiate the RBB010 from RBB012 variants by mass spec because of a point mutation in the V L sequence. C-terminal to the 3E10 subunit are the clevable linkers (Swivel, CTB) followed by a modified human HSP72 common to all 3 variants. The ITP and FGD peptides track this modified HSP72 and differentiate it from endogenous HSP72 in tissue extracts. The ITP peptide detects Fv-HSP72 regardless of the oxidative state of the tissues it is in. FGD has a methionine residue that can be oxidized and allows us to evaluate the relative oxidation level of our drug by setting the mass spectrometer to detect FGD peptide in three states of oxidation (methionine unoxidized, methionine sulfoxide and methionine sulfone).

Article Snippet: Rubicon Biotechnology has engineered nearly 20 variants of Fv-HSP72 to improve the therapeutic’s efficacy in a microenvironment that is mildly acidic , and enriched in reactive radicals .

Techniques: Mass Spectrometry, Mutagenesis, Sequencing, Modification, Residue

Journal: bioRxiv

Article Title: Rapid Intracellular Delivery of Human Heat Shock Protein 72 Inhibits Neurodegeneration and Oxidative Damage After a Traumatic Brain Injury

doi: 10.64898/2026.05.03.722564

Figure Lengend Snippet: A) Schematic of the CCI method. 100µg of cortical tissue extract from each of the rats were plated in triplicate in the wells of an ELISA plate and probed, as described in Methods, at a 1:2000 dilution with a rabbit monoclonal to the pTau S396 phosphorylation site followed by a goat anti-rabbit conjugated to horseradish peroxidase (GAR-HRP). TMB color development was stopped at 5min for this analysis. B) A scatter plot showing all of the triplicate results from both the ipsilateral (darker color) and contralateral (lighter color) hemispheres of each rat is a daunting task to read. The mean for the three contralateral absorbance values from Rat #35 were 327.15% greater than the mean for the other contralateral results; hence this data set was dropped from our analysis (circled in blue). C) To help visualize the data easier, the same results from each group of rats are presented as box plots with the ipsilateral results on the left and the contralateral on the right. Note that the graphs in (B) and (C) reveal global differences both between treatment groups and within groups (i.e. when comparing ipsi vs. contra). D) It is equally important to analyze biomarker differences between the ipsi- and contralateral hemispheres in each rat individually. This is accomplished by plotting the absorbance ratios against a black line denoting an equal distribution of signal in both hemispheres (ipsi / contra = 1). Analysis of the intra-hemispheric differences for each individual rat reveals a lower pTau S396 neurodegeneration signal level in the ipsilateral for rats treated with Fv-HSP72.

Article Snippet: Expanding on work by others showing HSP72 inhibiting brain lesions in mouse cortical impact models, we screened three Fv-HSP72 structural variants ( ) engineered by Rubicon Biotechnology in a rat CCI model simulating a moderate to severe impact.

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Biomarker Discovery

Journal: bioRxiv

Article Title: Rapid Intracellular Delivery of Human Heat Shock Protein 72 Inhibits Neurodegeneration and Oxidative Damage After a Traumatic Brain Injury

doi: 10.64898/2026.05.03.722564

Figure Lengend Snippet: Schematics of the 3 Fv-HSP72 variants tested. The ESG and YASYL peptides allow for detection of the 3E10 scFv subunit consisting of a variable light chain (V L ), linker, variable heavy chain (V H ), and a portion of a human constant heavy chain (CH1). These peptides also allow us to differentiate the RBB010 from RBB012 variants by mass spec because of a point mutation in the V L sequence. C-terminal to the 3E10 subunit are the clevable linkers (Swivel, CTB) followed by a modified human HSP72 common to all 3 variants. The ITP and FGD peptides track this modified HSP72 and differentiate it from endogenous HSP72 in tissue extracts. The ITP peptide detects Fv-HSP72 regardless of the oxidative state of the tissues it is in. FGD has a methionine residue that can be oxidized and allows us to evaluate the relative oxidation level of our drug by setting the mass spectrometer to detect FGD peptide in three states of oxidation (methionine unoxidized, methionine sulfoxide and methionine sulfone).

Article Snippet: Expanding on work by others showing HSP72 inhibiting brain lesions in mouse cortical impact models, we screened three Fv-HSP72 structural variants ( ) engineered by Rubicon Biotechnology in a rat CCI model simulating a moderate to severe impact.

Techniques: Mass Spectrometry, Mutagenesis, Sequencing, Modification, Residue