![In sickle cell mice, heart proteomic analysis reveals that H/R stress activates proinflammatory and profibrotic pathways. (A) Schematic diagram of the experimental plan used in this study. (B) Ingenuity pathway analysis (IPA) networks generated interrogating proteins identified as differentially expressed in SS hearts under H/R stress compared with AA under H/R. The following pathways were identified as affected by H/R in the hearts of SS mice vs healthy animals: (1) fibrosis of the heart; (2) hypertrophy of the heart; and (3) apoptosis of cardiomyocytes. (C) Immunoblot analysis using specific antibodies against phosphorylated Nrf2 (p-Nrf2) and Nrf2 in the hearts of AA and SS mice in normoxia (N) and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± standard error of the mean (SEM; n = 4). §P < .05 (compared with AA normoxia); ∗P < .05 (compared with normoxia by 1-way analysis of variance [ANOVA]). (D) Immunoblot analysis, using specific antibodies against phosphorylated FGF receptor (p-FGF-R), FGF-R, p-PDGF-R-B, and PDGFR-B, in the hearts of AA and SS mice. A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as mean ± SEM (n = 4). §P < .05 (compared with AA normoxia); ∗P < .05 (compared with normoxia by 1-way ANOVA). (E) Immunoprecipitation (IP) from the hearts of AA and SS mice, treated similar to panel D, using specific anti–phospho-tyrosine (PY) antibodies (IP: PY), revealed with specific anti–TGF-β receptor (TGF-β-Rec) antibody (75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel). GAPDH in whole cell lysate (WCL) is used as loading controls. One representative gel from 4 others with similar results is presented. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia by t test). TGF, transforming growth factor; Wb, Western blot.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/145/17/10.1182_blood.2024024768/2/blood_bld-2024-024768-gr1.jpeg?Expires=1748619144&Signature=tRXgGy8XNaU3gFV4veNyN-LjUC7gPrLOCsIdnsl6-NNiDn~nxI-CPBg7GTBvgIro5OE~1RF1sUPd2HXwEz0LibjdnoXlc4KQaO7zIVAC9Djl4rc1Go2TuSyw~nS6Ucs4NR6bkszFWQyJaqxm5EskwxEGOd8ayDLyyt1kv3pGSpwN1SSE2gxw4riwzTCZeyx2fBLvjYsS2tOhbqWWQdStZIt4q3LOMCdnITjyBUrtgMePPTniD3HkQbt~yR1wHODRuTpbKviDv-LrLYIdO-W-QMW~2V-V87-GrXHpqXJp5kWtToJczMmzZG7VCrtdHz0mXEAo~tlBDnsrrZseZnT9Ow__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
In sickle cell mice, heart proteomic analysis reveals that H/R stress activates proinflammatory and profibrotic pathways. (A) Schematic diagram of the experimental plan used in this study. (B) Ingenuity pathway analysis (IPA) networks generated interrogating proteins identified as differentially expressed in SS hearts under H/R stress compared with AA under H/R. The following pathways were identified as affected by H/R in the hearts of SS mice vs healthy animals: (1) fibrosis of the heart; (2) hypertrophy of the heart; and (3) apoptosis of cardiomyocytes. (C) Immunoblot analysis using specific antibodies against phosphorylated Nrf2 (p-Nrf2) and Nrf2 in the hearts of AA and SS mice in normoxia (N) and exposed to H/R: hypoxia (8% oxygen; 10 hours), followed by reoxygenation (21% oxygen; 3 hours). A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as means ± standard error of the mean (SEM; n = 4). §P < .05 (compared with AA normoxia); ∗P < .05 (compared with normoxia by 1-way analysis of variance [ANOVA]). (D) Immunoblot analysis, using specific antibodies against phosphorylated FGF receptor (p-FGF-R), FGF-R, p-PDGF-R-B, and PDGFR-B, in the hearts of AA and SS mice. A total of 75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel. GAPDH serves as protein loading control. One representative gel from 4 with similar results is shown. Densitometric analysis of immunoblots is shown (right). Data are presented as mean ± SEM (n = 4). §P < .05 (compared with AA normoxia); ∗P < .05 (compared with normoxia by 1-way ANOVA). (E) Immunoprecipitation (IP) from the hearts of AA and SS mice, treated similar to panel D, using specific anti–phospho-tyrosine (PY) antibodies (IP: PY), revealed with specific anti–TGF-β receptor (TGF-β-Rec) antibody (75 μg/μL of protein loaded on an 8% T, 2.5% C polyacrylamide gel). GAPDH in whole cell lysate (WCL) is used as loading controls. One representative gel from 4 others with similar results is presented. Data are presented as means ± SEM (n = 4). ∗P < .05 (compared with normoxia by t test). TGF, transforming growth factor; Wb, Western blot.
