Fig 2: A) Histograms of differential protein expression in cardiac tissue of old adult (22 months, gray bars) and senescent (30 months, black bars) rats, as detected by 2-D DIGE analysis. Proteins significantly altered (ANOVA and Tukey, p < 0.01) were expressed as a ratio of spot volume variations in old adult and senescent rats versus young (6 months) control, respectively. In rat hearts, 6 stress response and 4 other proteins were identified: Gstp1, glutathione S-transferase P; Hspa8, heat shock cognate 71 kDa protein; Mpst, 3-mercaptopyruvate sulfurtransferase; Pdia3, protein disulfide-isomerase A3; Prdx2, peroxiredoxin-2; Rnh1, ribonuclease inhibitor; Anxa5, annexin A5; Serpina1, alpha-1-antiproteinase (2 proteoforms); Serpina3k, serine protease inhibitor A3 K; Serpina3l, serine protease inhibitor A3L. B) Validation of Hspa8 protein by immunoblotting in young, old adult and senescent rat cardiac muscles. Statistical analysis was performed by Student’s T-Test (n = 3; * p < 0.05). Data were normalized against the total amount of loaded proteins stained with Sypro Ruby.

Fig 3: PKG regulates CHIP-chaperone interaction and post-translational stability.a Structural model of binding region between Hsc70 lid motif and the TPR region in CHIP. The location of S20 in reference to opposing lysine residues K597 and K601 in Hsc70 is shown. b Upper: example immunoprecipitation using Hsc70 as bait, and detecting Myc-tagged WT, SE, or SA CHIP in both control and SI conditions. There is minimal Hsc70-CHIP co-precipitation when CHIP-SA is expressed. Lower: summary data, n = 3 biological replicates/group, 1WANOVA for each condition, Holms-Sidaks mct: *p = 1.2 × 10−4, †0.003, ‡0.0005, §0.02. c Immunoprecipitation gels and summary data for Hsc70 and CHIP interaction in vivo with and without MI in mice with mPDE5oe and littermate controls (CON). Hsc70 is bait, and antibody detection using CHIP and Hsc70. N = 4 biological replicates/group, Kruskal-Wallis test, Dunns mct. P values in figure. Source data are provided as a Source Data file. Individual data and mean ± SEM shown in each summary panel.

Fig 4: Isolation and verification of exosomes derived from KG-1a cells and BMSCs. (A) Abundance of TSG101, HSPA8, and Alix proteins in exosomes derived from KG-1a cells and BMSCs. WB analysis was performed to detect protein abundance. (B) Size distribution of exosomes secreted by KG-1a cells and BMSCs. Exosome sizes were analyzed using NTA. BMSCs: Bone marrow-derived mesenchymal stem cells; TSG101: Tumor susceptibility 101; HSPA8: Heat shock protein family A (Hsp70) member 8; Alix: Programmed cell death 6 interacting protein; WB: Western blot; NTA: Nanoparticle tracking analysis; exo: Exosomes.

Fig 5: GP5 disrupts the interaction between LAMP2A and HSC70 by competitively binding to LAMP2A. (A) PAMs were infected with PRRSV at an MOI of 1 for 36 h, and a co-IP assay was performed by the anti-PRRSV-GP5 antibody. GP5-interacting host proteins were eluted and analyzed on SDS-PAGE followed by silver staining. *: HSC70 or LAMP2A. (B, D) HEK293T cells were cotransfected with Flag-GP5 and Myc-HSC70 (B), or Flag-GP5 and Myc-LAMP2A (D) for 24 h. The cell lysates were immunoprecipitated with anti-Flag IgG beads and subjected to immunoblotting using the indicated antibodies. (C, E) 3D4/21 cells were cotransfected with GFP-GP5 and Flag-HSC70 (C), or GFP-GP5 and mCherry-LAMP2A (E) for 24 h. Cells were immunostained with anti-Flag mAb and observed under a confocal microscope. (F, G) PAMs (F) and MARC-145 cells (G) were infected with PRRSV at an MOI of 1 for 36 h, and a co-IP assay was performed by the anti-PRRSV-GP5 antibody. GP5-interacting host proteins were eluted and analyzed on immunoblotting. (H) Schematic representation of GP5 mutants. The CMA motif was marked in red. The mutant regions were marked in yellow. (I) HEK293T cells were cotransfected with vectors expressing GP5 mutants and Myc-HSC70 for 24 h. The cell lysates were immunoprecipitated with anti-Flag IgG beads and subjected to immunoblotting using the indicated antibodies. (J) HEK293T cells were transfected with Flag-GP5 (2 µg) and increasing amounts of Myc-HSC70 (0–2 μg) for 24 h. The cell lysates were then subjected to immunoblotting using the indicated antibodies. (K, L) HEK293T cells were cotransfected with Flag-LAMP2AΔTail and Myc-GP5 (K) or Flag-LAMP2AΔTail and Myc-HSC70 (L) for 24 h. The cell lysates were subjected to analysis of Flag-precipitation and immunoblotting analysis. (M, N) HEK293T cells were cotransfected with Flag-LAMP2A, Myc-HSC70, and HA-GP5 for 24 h (M). HEK293T cells were cotransfected with Flag-LAMP2A and Myc-GP5 for 24 h (N). The cell lysates were subjected to analysis of Flag-precipitation and immunoblotting analysis. (O) 3D4/21 cells were transfected with Myc-GP5, Flag-HSC70, and mCherry-LAMP2A for 24 h. Cells were immunostained with anti-Myc pAb and anti-Flag mAb and observed under a confocal microscope. WCL: whole cell lysate. The scale bar indicates 5 µm. Error bars: mean ± SD of 3 independent tests. student’s t-test; ***P < 0.001 compared to control.