Fig 1: Recombinant HSPA9 interaction with overlapping peptides derived from E protein domain I (DI) and extended domain II (DII). Peptides were allowed to bind to HSPA9 (4 µg/mL) and tested for their HSPA9 binding capability by using indirect enzyme-linked immunosorbent assay. OD, optical density.
Fig 2: Identification of the HSPA9 binding domains in E protein. (A) Determination of HSPA9 binding domains in E protein by Western blot. The membrane containing transferred HSPA9 protein was incubated with purified recombinant protein of PBST (lane 1), DIII (lane 2), DII (lane 3), or DI (lane 4) and subsequently detected by adding anti-TMUV serum; lane M, molecular weight marker. (B) SDS-PAGE analysis of co-immunoprecipitation assay. The purified recombinant DI (lane 1; black arrow), DII (lane 2; black arrow), or DIII (lane 4) proteins were incubated with purified recombinant HSPA9 protein followed by incubation with anti-HSPA9 antibody. The co-immunoprecipitated complexes were then separated by SDS-PAGE. As controls, purified recombinant DI (lane 3), DII (lane 5), or DIII (lane 6) proteins were incubated without purified recombinant HSPA9 protein followed by incubation with anti-HSPA9 antibody. Lane M, molecular weight marker. PBST, phosphate buffered saline with Tween 20 (NaCl 137 mM, KCl 2.7 mM, Na2HPO4 4.3 mM, KH2PO4 1.4 mM, pH to 7.4, with 0.05% Tween 20); TMUV, Tembusu virus; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DI, E protein domain I; DII, extended domain II; DIII, globular domain III.
Fig 3: Identification of the minimal HSPA9-binding motif by using truncated peptides. Truncated peptides were probed for reactivity with recombinant HSPA9 by Dot blot assay. Full-length E protein and DIII protein were used as positive and negative controls, respectively. (A) Identification of the HSPA9-binding motif by using truncated peptides. (B) Validation of minimal HSPA9-binding motif by Dot blot assay.
Fig 4: Sevoflurane upregulates IP3R and activates endoplasmic reticulum stress and the caspase-related apoptotic pathway in isolated hippocampal neurons. (A) Representative western blot of p-IP3R, t-IP3R, MFN1, MFN2, GRP75, p-PERK, ATF4, CHOP, cleaved caspase-9, cleaved caspase-3, Bcl-2, and Bax. (B) Representative histogram of the relative expression of p-IP3R, t-IP3R, MFN1, MFN2, GRP75, p-PERK, ATF4, CHOP, cleaved caspase-9, cleaved caspase-3, Bcl-2 and Bax. Data are presented as mean ± SD (n = 5 in each group). Compared with the Control group, *p < 0.05; compared with the Sev group, #p < 0.05.
Fig 5: Sevoflurane upregulates p-IP3R and activates endoplasmic reticulum stress in aged rats. (A) Representative western blot of p-IP3R, t-IP3R, MFN1, MFN2, GRP75, p-PERK, ATF4 and CHOP. (B) Representative histogram of the relative expression of p-IP3R, t-IP3R, MFN1, MFN2, GRP75, p-PERK, ATF4, and CHOP. (C) Representative images of immunohistochemistry of p-IP3R in CA1 (scale bar = 50 µM). (D) Representative histogram of positive cell rate of p-IP3R. Data are presented as mean ± SD (n = 5 in each group). Compared with the Control group, *p < 0.05; compared with the Sev group, #p < 0.05.
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