syntaxin 4 crispr cas9 ko plasmid h  (Santa Cruz Biotechnology)

Journal: Nature Metabolism

Article Title: Feeding-regulated glycogen metabolism drives rhythmic liver protein secretion

doi: 10.1038/s42255-026-01453-8

Figure Lengend Snippet: a , Experimental design (created with BioRender.com ). WT mice were maintained under a 12 h light–dark cycle and exposed to NR feeding. Liver tissue was collected every 3 h over two consecutive days ( n = 16 (8 timepoints × 2 biological replicates)). b , Heatmap representation of the rhythmic microsomal proteome (adjusted P < 0.1) analysed across two independent days (day 1 and day 2). Data are row-standardized. c , Number of rhythmic microsomal liver proteins as a function of minimal amplitude. The colour code represents different false discovery rate (FDR) values. d , Phase distribution of rhythmic microsomal liver proteins. The colour code represents different FDR values. e , Scatterplot showing the relationship between acrophase and amplitude of rhythmic microsomal proteins. f , Examples of two rhythmic microsomal proteins confirmed by western blot: STX4 and ARFGAP1 (top). Quantification of western blot analysis (bottom). LC, loading control. MS: n = 16, 8 timepoints × 2 biological replicates; WB: n = 23, 8 timepoints × 3 biological replicates, except ZT21 (two biological replicates). g , Gene set enrichment analysis of rhythmic proteins in the secretory pathway. Adjusted P = 0.05, indicated by a dashed line. h , Phase distribution of microsomal rhythmic proteins associated with the ER and GA (statistical difference in phase evaluated by Kolmogorov–Smirnov test, P = 2.2 × 10⁻¹⁶). i , Scatterplot showing the relationship between acrophase and amplitude of rhythmic ER-associated (left) and GA-associated (right) proteins. j , Representative images (left) and quantitative analysis (right) of electron microscopy imaging of mouse livers at one timepoint during the day (ZT4) and night (ZT16) under NR feeding ( n = 4 biological replicates with GA ZT16 ( n = 3). ER and GA are highlighted in orange and blue, respectively. N, nucleus; mt, mitochondria. Each colour in the graphs represents an independent biological replicate. Several independent liver areas per biological replicate were analysed. k , Heatmap representation of rhythmic proteins involved in protein glycosylation, categorized by function. Data are row-standardized. l , Scatterplot showing the relationship between acrophase and amplitude of N -glycans on the indicated protein. Non-complex (oligomannose) and complex glycosylation modifications. m , Phase difference between rhythmic oligomannose (top) and complex (bottom) N -glycans in mouse liver (two-tailed Kolmogorov–Smirnov test for the difference in phase, P = 0.011). n , Temporal profiles of the microsomal proteins and N -glycans at indicated sites of RAB2 and H2-K1. Temporal profiles of microsomal proteins, oligomannose and complex N -glycans at indicated positions ( n = 16 (8 timepoints × 2 biological replicates)). Data are displayed as means; error bars, s.e.m. Tukey boxplots show the median and interquartile range, whiskers extend to the most extreme values within 1.5× the interquartile range and outliers are shown as individual points. A detailed description of the statistical analysis is available in Source Data Fig. . See also Extended Data Fig. and Supplementary Tables and for related data.

Article Snippet: Primary antibodies were used at the following dilutions: 1:1,000 for ATF4 (Cell Signaling Technologies, 11815), ARFGAP1 (Cell Signaling Technologies, 14608), Phospho-RPS6 (Cell Signaling Technologies, 2211), Total-RPS6 (Cell Signaling Technologies, 2217), GABARAPL1 (Genetex, GTX132664) and ConA Lectin (Vector Laboratories, B-1005) and 1:2,000 for STX4 (ProteinTech, 14988-1-AP).

Techniques: Western Blot, Control, Electron Microscopy, Imaging, Glycoproteomics, Two Tailed Test

Journal: bioRxiv

Article Title: Dual Ligand Cooperation at the Plasma Membrane Drives Transport of Engineered Small Extracellular Vesicles Across Brain Endothelial Cells

doi: 10.64898/2026.01.21.700773

Figure Lengend Snippet: sEVs-HA-Tf accumulate at the basolateral membrane of BECs. (a) Scheme illustrating the trancystosis steps of sEVs or sEVs-HA-Tf in BECs. (b.1) Expression of syntaxin-4 in human BECs along the z-axis. xy-slice at the level of the basolateral (z-stack:2/32) and apical (z-stack:17/32) membranes. Scale bar: 40 µm. (b.2) Number of Syntaxin-4 foci along the z-axis. (c.1) Representative image of Transferrin-594 conjugate, acquired at the bottom most plane of the glass slide, on STED mode using a single point scanning confocal Stellaris 8 (Leica) microscope. (c.2) Representative images of EVs, EVs-HA and EVs-HA-Tf, acquired with similar settings. Scale bar: 1 µm. (c.3) Measured area (μm2) of the encircled organelle structures depicted in c.1 and c.2. Results are expressed as mean±SEM (3 fields analyzed per experimental condition across 105-135 cells in a single experiment). (d.1) Overview of Transferrin and HSA co-localization with syntaxin-4 at the basolateral membrane. Arrowheads point to two-channel overlapping pixels. Scale bar: 20 µm. (d.2) Co-localization of transferrin and HSA with the t-SNARE protein syntaxin-4. Results are expressed as mean±SEM (5 fields analyzed per experimental condition across 175-225 cells in a single experiment). Results are given by the Mander’s co-localization coefficient M1, normalized to the control. Statistical analysis was performed by a Mann–Whitney test (**: p ≤ 0.01). In b.1, b.2, d.1 and d.2, images were acquired in a LSM710 confocal microscope (Zeiss). (e.1) Representative images of sEVs, sEVs-HA, and sEVs-HA-Tf interaction with Syntaxin-4, acquired on STED mode at the plane closest to the glass slide (corresponding to the basolateral side of the cell). Scale bar: 5 µm. Inset: Higher magnification images of EVs in contact with Syntaxin-4. Scale bar: 1 µm. (e.2) Co-localization of sEVs with syntaxin-4, given by the number of sEVs carrying organelles that colocalize with Syntaxin-4. Colocalization is defined as all Syntaxin-4 objects distancing ≤200 nm from sEVs carrying organelles. Results were normalized to the control. (f.) Number of basolateral sEVs carryring organelles, normalized to the control. Results in e.2 and f. are expressed as mean±SEM (11-13 fields analyzed per experimental condition across 25-50 cells in 2 independent experiments). An independent experiment is defined as a separate bioconjugation reaction performed using sEVs obtained from 2-3 isolations derived from pooled plasma of 4-6 individual donors. Statistical analyses were performed by One-way ANOVA, followed by Tukey’s multiple comparisons test (p<0.05).

Article Snippet: To evaluate the expression of the t-SNARE protein Syntaxin-4 on the basolateral membrane, cells were seeded on type-I collagen coated ibidi μ-Slide 15 wells 3D, at a density of 50,000 cells/cm 2 , for 2 days, fixed and immunostained for Syntaxin-4 1:100 (R&D Systems, MAB7894), followed by incubation with Alexa-fluor 488 donkey anti-mouse 1:800 secondary antibody in 1% (w/v) BSA for 1 h in the dark at room temperature.

Techniques: Membrane, Expressing, Microscopy, Control, MANN-WHITNEY, Derivative Assay, Clinical Proteomics

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-Stx4 rabbit monoclonal antibody , Proteintech , Cat# 14988-1-AP; RRID:AB_2286910.

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