anti stx3 rabbit polyclonal antibody  (Proteintech)

Journal: iScience

Article Title: Legionella employs the multimodal ubiquitination of Sec22b to modulate SNARE pairing

doi: 10.1016/j.isci.2025.114341

Figure Lengend Snippet: Ubiquitination of Sec22b promotes non-canonical SNARE pairings with Stx3 and Stx4 (A) HEK293T-FcγRII cells stably expressing FLAG-Sec22b were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h. FLAG-Sec22b derivatives were immunoprecipitated from the cell lysates at the indicated time points using anti-FLAG beads. The cell lysates (input) and the immunoprecipitated proteins (IP: FLAG) were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The intensity values of Stx3 protein bands were quantified using the ChemiDoc System with Image Lab software (Bio-Rad), normalized to the value of the no-infection condition (no bacteria), and displayed below the immunoblotting data. (B) HEK293T-FcγRII cells stably expressing FLAG-Sec22b S137A were infected with the indicated L. pneumophila strains at an MOI of 80 for 1 h, and analyzed as described in (A). The intensity values of Stx3 protein bands were quantified as described in (A) and displayed below the immunoblotting data. (C) HEK293T-FcγRII cells stably expressing FLAG-Sec22b or FLAG-Sec22b S137A were infected with the wild-type strain Lp01 at an MOI of 80 for 1 h, and analyzed as described in (A). (D) Intensity values of Stx3, Stx4, and Stx18 protein bands from (C) quantified as described in (A), normalized to the respective Syntaxin values bound to Sec22b WT. Values represent the mean ± SEM from three independent experiments. ns, not significant. ∗ p < 0.05, ∗∗∗∗ p < 0.0001.

Article Snippet: Anti-Stx3 rabbit polyclonal antibody (15556-1-AP) and anti-Stx4 rabbit monoclonal antibody (14988-1-AP) were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Infection, Immunoprecipitation, SDS Page, Western Blot, Software, Bacteria

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A SNAP29, SNAP23, STX3, SEC22B, and FKBP5 co-immunoprecipitation (SKA2 IP) and whole cell extract (WCE) in hippocampus (HIP), prefrontal cortex (PFC) and amygdala (AMY) samples of mice ( n = 8). B HIS pull down assay (replicated in 3 independent in vitro experiments). DDK(Flag)-tagged SNAP23, SNAP29, Syntaxin3 or Syntaxin4 was incubated with purified magnetic beads-HIS-tagged SKA2 or magnetic beads-HIS protein alone. After incubation, bead bound proteins were eluted at room temperature (RT) or at 95 °C and subjected to western blot analysis using antibodies against HIS and FLAG. Input lane contains HIS alone (left) or HIS-tagged SKA2 (right). C – M SIM-A9 cells transfected with SKA2, FKBP5 or their respective controls, were harvested 24 h later. After immunoprecipitation (IP) of protein complexes, input and co-IP proteins were quantified by western blotting. C , F , I , K Representative blots of ( D , E , G , H , J , L , M ). Graphs display quantification of SNAP29/SEC22B, STX3/SEC22B, SKA2/SNAP29, FKBP5/SEC22B protein association after SEC22B or SNAP29 IP (unpaired two tailed t-test: ( D ) t 6 = 8.945, p < 0.0001, ( E ) t 6 = 12.94, p < 0.0001, ( G ) t 6 = 6.056, p = 0.0009, ( H ) t 6 = 5.554, p = 0.0014; one-way ANOVA: ( J ) F 2, 9 = 17.28, p = 0.0008, Tukey’s post hoc test: ctrl vs. FKBP5-OE, p = 0.0743, ctrl vs. FKBP5-KO, p = 0.0218, FKBP5-OE vs. FKBP5-KO, p = 0.0006; unpaired two tailed t-test: ( L ) t 6 = 10.27, p < 0.0001, ( M ) t 6 = 8.140, p = 0.0002; n = mean derived from four independent in vitro experiments). * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: Primary antibodies used : FKBP5 (1:1000, Bethyl, A301-430A), FKBP5 (1:1000, Cell Signaling Technology, #12210), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signaling Technology, #67657), SEC22B (1:1000, Abcam, ab181076), SKA2 (1:1000, Thermo Fisher, PA5-20818), SKA2 (1:500, Millipore-Sigma, SAB3500102), SKA1 (1:1000, Thermo Fisher, PA5-20817), SKA3 (1:1000, Thermo Fisher, PA5-20819), GSDMD (1:1000, Cell Signaling Technology, 39754), VCP (1:10000, Abcam, Ab11433), NEK7 (1:50, Abcam, Ab133514), NLRP3 (1:50, Cell Signaling Technology, 15101), HIS (1:5000, Cell Signaling Technology, #12698), FLAG M2 (1:10000, Sigma, A8592).

Techniques: Immunoprecipitation, Pull Down Assay, In Vitro, Incubation, Purification, Magnetic Beads, Western Blot, Transfection, Co-Immunoprecipitation Assay, Two Tailed Test, Derivative Assay

Journal: Nature Communications

Article Title: SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration

doi: 10.1038/s41467-024-46953-x

Figure Lengend Snippet: A , B IL-1β release measured via ELISA from supernatants of SIM-A9 cells 24 h after manipulation of SKA2 and/or FKBP5 expression, and following overnight LPS (100 ng/mL) and treatment with LLOMe (0.25 mM) for 3 h (unpaired two tailed t-test: (A) t 4 = 11.99, p = 0.0003; one-way ANOVA: B F 3, 8 = 158.6, p < 0.0001; Tukey’s post hoc test: ctrl vs. SKA2-OE, p = 0.0384, ctrl vs. FKBP5-OE, p < 0.0001, SKA2-OE vs. FKBP5-OE, p < 0.0001, FKBP5-OE vs. SKA2 + FKBP5 OE, p < 0.0001; n = mean derived from three independent in vitro experiments). C Schematic overview of the SA pathway with SKA2 and FKBP5. The cargo receptor TRIM16, together with SEC22B, transfers molecular cargo (e.g., IL-1β) to the autophagy-related LC3B-positive membrane carriers. SEC22B, now acting as an R-SNARE on the delimiting membrane facing the cytosol, carries out fusion at the plasma membrane in conjunction with the Q bc -SNAREs, SNAP23 and SNAP29 (SNAP23/29), and one of the plasma membrane Q a -SNAREs, STX3 or STX4 (STX3/4), thus delivering IL-1β to the extracellular milieu, where it exerts its biological functions. FKBP5 acts as a positive regulator of SA by enhancing TRIM16-SEC22B complex formation as well as autophagosome-plasma membrane fusion via the SNARE-protein complex assembly. In contrast, SKA2 inhibits the SNARE-protein complex formation during vesicle-plasma membrane fusion, thereby acting as gatekeeper of SA. D , E Schematic overview of in vivo microdialysis and the experimental design and timeline; each sample was collected over 30 min indicated by the light gray lines. Quantifications of IL-1β, determined by capillary-based immunoblotting from in vivo medioprefrontal cortex microdialysis of C57Bl/6NCrl mice injected intraperitoneally with ULK1 inhibitor (ULK1i, an autophagy inhibitor) or saline ( F ; repeated measures two-way ANOVA, time × treatment interaction: F 5, 30 = 7.064, p = 0.0002; Šidák’s multiple comparisons post hoc test, post-FS-1: p = 0.0084; n = 4 mice per group) as well as of wild type (WT) and global Fkbp5 knockout mice ( G ; repeated measures two-way ANOVA, time × genotype interaction: F 5, 30 = 34.15, p < 0.0001; Šidák’s multiple comparisons post hoc test: FS: p = 0.009, post-FS-1: p = 0.0163, post-FS-2: p = 0.0294; n = 4 mice per group). FS foot shock. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. Data are presented as mean + SEM. Source data are provided as a file.

Article Snippet: Primary antibodies used : FKBP5 (1:1000, Bethyl, A301-430A), FKBP5 (1:1000, Cell Signaling Technology, #12210), ACTIN (1:5000, Santa Cruz Biotechnology, sc-1616), GAPDH (1:8000, Millipore CB1001), SNAP29 (1:1000, Sigma, SAB1408650), SNAP23 (1:1000, Sigma, SAB2102251), STX3 (1:1000, Sigma, SAB2701366), STX4 (1:1000, Cell Signaling Technology, #67657), SEC22B (1:1000, Abcam, ab181076), SKA2 (1:1000, Thermo Fisher, PA5-20818), SKA2 (1:500, Millipore-Sigma, SAB3500102), SKA1 (1:1000, Thermo Fisher, PA5-20817), SKA3 (1:1000, Thermo Fisher, PA5-20819), GSDMD (1:1000, Cell Signaling Technology, 39754), VCP (1:10000, Abcam, Ab11433), NEK7 (1:50, Abcam, Ab133514), NLRP3 (1:50, Cell Signaling Technology, 15101), HIS (1:5000, Cell Signaling Technology, #12698), FLAG M2 (1:10000, Sigma, A8592).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test, Derivative Assay, In Vitro, Membrane, Clinical Proteomics, In Vivo, Western Blot, Injection, Saline, Knock-Out