Fig 1: TNFAIP1 knockdown ameliorates postoperative cognitive impairment, which is abrogated by SNAP25 knockdown. A Experimental design. Two weeks after AAV9 injection, mice were subjected to laparotomy under isoflurane anesthesia. The OFT and FCT were performed from the third to fifth day. The hippocampus of different groups was harvested after behavioral assessment. The total moving distance (B) time spent in the center region (C) in the OFT. D Freezing response of mice was assessed by contextual fear conditioning as the proportion of immobility (n = 12). **P < 0.01, one-way ANOVA test. E H&E (400 × , bar: 50 μm) and TUNEL staining (400 × , bar: 50 μm) of hippocampal CA1 samples following the indicated treatments. TUNEL-positive cells are shown in red, and nuclei are shown in blue. F The Percentage of TUNEL-positive cells in the hippocampi of mice (n = 3). **P < 0.01, one-way ANOVA test. G Gel images of mitophagy- and pyroptosis-related proteins in the hippocampi microinjected with the indicated viruses. GAPDH served as a loading control (left panel). Quantification results normalized to GAPDH were represented as mean ± SEM (right panel, n = 4). *P < 0.05, **P < 0.01, one-way ANOVA test. The concentration of IL-1β and IL-18 was measured by ELISA in the serum (H-I) and hippocampi (J-K) of mice following the indicated treatments (n = 4). *P < 0.05, **P < 0.01, one-way ANOVA test
Fig 2: Increased expression of the synaptic protein PSD-95 in neuroserpin-deficient mice. Representative Western blots of hippocampal extracts from three different animals separated by SDS-PAGE and analyzed with antibodies against PSD-95 (postsynapse) and synaptophysin, SNAP25, and synapsin-I (presynapse). Band intensity was normalized to β-actin expression. Relative expression is presented (AU, arbitrary units), wild-type was set to 1 (mean ± SD; n = 3–4; P = 0.027 for PSD-95; P = 0.100 for synaptophysin; P = 0.881 for SNAP25; P = 0.494 for synapsin-I).
Fig 3: TNFAIP1 catalyzes K48-linked polyubiquitination of SNAP25 at K69. A Ubiquitination analysis of SNAP25 in lysates from HEK293T cells transfected with Myc-SNAP25, HA-Ub, and full-length Flag-tagged TNFAIP1 or its truncations. B Co-transfection of Flag-TNFAIP1 with Myc-SNAP25 and WT or mutant HA-tagged ubiquitin in HEK293T cells followed by ubiquitination assays of exogenous SNAP25. C Diagram illustrating the predicted ubiquitination sites of SNAP25 (colored in pink, top), and ubiquitination assays of SNAP25 in lysates from HEK293T cells transfected with Flag-TNFAIP1, HA-Ub, and WT or mutant Myc-tagged SNAP25 (bottom). D Western blot analysis of SNAP25 protein levels in HT22 cells treated with MG132 (50 nM, 4 h) and Flag-TNFAIP1. E Quantification results normalized to GAPDH are presented as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, one-way ANOVA test. F Immunoblot analysis of SNAP25 in HT22 cells transfected with Flag-TNFAIP1 in the absence or presence of CHX (10 μg/mL). G The relative SNAP25 level for each time point was quantified as mean ± SEM (n = 3). **P < 0.01, two-way ANOVA test. H HT22 cells transiently transfected with full-length Flag-tagged TNFAIP1 or its truncations were treated with 10 μg/mL CHX for 0, 3, and 6 h. (I) Quantification of SNAP25 levels relative to GAPDH was shown as mean ± SEM (n = 3). **P < 0.01, 97–216 vs. FL; #P < 0.05, ##P < 0.01, 207–316 vs. FL, two-way ANOVA test
Fig 4: FGF21 promoted insulin secretion along with up‐regulation of SNARE proteins. Sixteen‐week‐old db/db mice and their lean controls were intraperitoneally injected with AAV‐F21 or AAV‐GFP and killed 4 weeks later. (A) The plasma insulin levels were measured immediately following the killing. (B) Static glucose‐stimulated insulin secretion (GSIS) in isolated islets was measured at 1 h after 16.7 mmol/L glucose stimulation. (C–E) Quantitative PCR analysis of STX1, SNAP25 and VAMP2 in isolated islets. (F–H) Western blot analysis of STX1, SNAP25 and VAMP2 in isolated islets. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs the lean controls; *P < 0.05 and **P < 0.01 vs db/db mice treated with AAV‐GFP. (A) n = 5. (B–H) n = 3‐5
Fig 5: SNAP25 overexpression rescues the defective mitophagy and excessive pyroptosis phenotypes induced by TNFAIP1 overexpression. A HT22 cells were transfected with indicated plasmids and treated with 3% isoflurane (30 min) and 1 μg/mL LPS (12 h), followed by incubation with 10 mM DCFH-DA for 20 min. The intracellular ROS level was reflected by the MFI of DCFH-DA (n = 3). **P < 0.01, one-way ANOVA test. B Flow cytometry analysis of double staining with Annexin V-FITC/PI under Iso + LPS conditions in HT22 cells overexpressing indicated plasmids. C Quantification of pyroptotic cells with annexin V-FITC/PI double-positive staining (n = 3). **P < 0.01, one-way ANOVA test. D Gel images of mitophagy- and pyroptosis-related proteins in Iso + LPS-treated HT22 cells transfected with indicated plasmids. GAPDH served as a loading control. E Quantification results normalized to GAPDH were presented as mean ± SEM (n = 4). *P < 0.05, **P < 0.01, one-way ANOVA test
Supplier Page from Abcam for Anti-SNAP25 antibody