Journal: iScience

Article Title: Mutation T9I in Envelope confers autophagy resistance to SARS-CoV-2 Omicron

doi: 10.1016/j.isci.2025.112974

Figure Lengend Snippet: E T9I has increased affinity to autophagosome-associated proteins (A) Principal component analysis of the differential interactome data , the individual replicates are separated (black: GFP controls, Green: E T9 pulldown, Purple: E T9I pulldown) (B) Volcano plot of the differential interactome analysis showing enriched proteins in E T9I pulldown versus the p value (-log P). Five highly significantly enriched proteins are highlighted in red and via labels. (C–G) Quantification of proximity ligation assays between transiently expressed SARS-CoV-2 E variants 30 h post transfection in HeLa cells and endogenous SNX12, STX12, TMEM87B, ABCG2 and TAB1, as indicated. Representative images depicted. PLA signal, red. Scale Bar, 10μm. DAPI, nuclei (blue). Lines represent the mean of N = 18–59 (individual cells) ±SEM. (H) Quantification of autophagosome levels by flow cytometry in HEK293T autophagy reporter cells (HEK293T-GL) transiently expressing StrepII-tagged SARS-CoV-2 E variants (48 h post transfection) and depleted of indicated proteins by siRNA. Bars represent the mean of N = 3 (biological replicates) ±SEM. Student’s t test with Welch’s correction. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: Polyclonal rabbit anti-STX12 Antibody (PLA 1:100) (WB 1:500) (IP) , Proteintech , Cat#14259-1-AP; RRID: AB_2198222.

Techniques: Ligation, Transfection, Flow Cytometry, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Pulmonary mitochondrial DNA release and activation of the cGAS-STING pathway in Lethal Stx12 knockout mice.

doi: 10.1186/s12964-025-02141-y

Figure Lengend Snippet: Fig. 1 STX12 deficiency induces mitochondrial membrane potential defects in zebrafish. A A time-course plot of percent survival in the control vs. Stx12 morphants for 3 days. dpf, days postfertilization; hpf, hours postfertilization. B Representative images of five selected stages of zebrafish embryos. Scale bar: 100 μm. C qRT-PCR for five embryo development stages (0.2 hpf, 1 hpf, 2 hpf, 3.7 hpf and 6 hpf) demonstrating different expression patterns of Stx12 during embryonic development. D Representative images of TMRM staining and AO staining in control, E4I4-MO and ATG-MO zebrafish. Treatment window: 2 hpf-3.7 hpf; Stage of image: 3.7 hpf. TMRM staining: 1 μM; AO staining: 5 μg/mL. n = 10, Scale bar: 100 μm. E Quantification of the relative TMRM fluorescence intensity. F Quantification of apoptosis particle number in AO staining of control, E4I4-MO and ATG-MO zebrafish. Image analysis was performed using ImageJ software, with n = 10 per group. G Representative images of TMRM staining and AO staining after CCCP treatment. Stage of Image: 3.7hpf. Scale bar: 300 μm. Data are presented as mean ± SEM; statistical significance was assessed by Student’s t-test

Article Snippet: The primary antibodies used included the following: rabbit anti-STX12 monoclonal antibody (1:1000, custom-made), mouse anti-β-actin (1:5000; Proteintech, #60,008–1-Ig), mouse anti-GAPDH (1:20,000, Proteintech, #60,004–1-Ig), anti-OXPHOS antibody (1:1000; Abcam, #ab110413, Cambridge, UK), anti-cGAS (1:1000; Cell Signaling Technology, #31,659, Danvers, MA, USA), anti-TBK1(1:1000, Cell Signaling Technology, #3504S), anti-pTBK1(1:1000; Cell Signaling Technology, # 5483S), anti-IRF3(1:1000; Proteintech, #11,312–1-AP), antipIRF3(1:1000; Cell Signaling Technology, #29,047), antiIL-6 (1:1000; Abcam, #ab229381), anti-S100A9 (1:1000; Boster, #PB0718, Wuhan, China).

Techniques: Membrane, Control, Quantitative RT-PCR, Expressing, Staining, Fluorescence, Software

Journal: Cell communication and signaling : CCS

Article Title: Pulmonary mitochondrial DNA release and activation of the cGAS-STING pathway in Lethal Stx12 knockout mice.

doi: 10.1186/s12964-025-02141-y

Figure Lengend Snippet: Fig. 6 Schematic of systemic immune-inflammation in STX12-KO mice. The ablation of STX12 leads to decreased mitochondrial membrane potential (MMP), reduced expression levels of mitochondrial complex subunits, and the release of mitochondrial DNA (mtDNA). Then, mtDNA release activates the cGAS-STING-pTBK1-pIRF3 pathway, subsequently triggering Type I interferon response and downstream interferon-stimulated genes (ISGs) and cytokines in lung tissue. Additionally, cytokines release and neutrophil infiltration mutually enhance each other, resulting in an amplified cascade of hyperinflammation, referred to as “cytokine storm”, which potentially contributes to the mortality observed in Stx12 knockout mice

Article Snippet: The primary antibodies used included the following: rabbit anti-STX12 monoclonal antibody (1:1000, custom-made), mouse anti-β-actin (1:5000; Proteintech, #60,008–1-Ig), mouse anti-GAPDH (1:20,000, Proteintech, #60,004–1-Ig), anti-OXPHOS antibody (1:1000; Abcam, #ab110413, Cambridge, UK), anti-cGAS (1:1000; Cell Signaling Technology, #31,659, Danvers, MA, USA), anti-TBK1(1:1000, Cell Signaling Technology, #3504S), anti-pTBK1(1:1000; Cell Signaling Technology, # 5483S), anti-IRF3(1:1000; Proteintech, #11,312–1-AP), antipIRF3(1:1000; Cell Signaling Technology, #29,047), antiIL-6 (1:1000; Abcam, #ab229381), anti-S100A9 (1:1000; Boster, #PB0718, Wuhan, China).

Techniques: Membrane, Expressing, Amplification, Knock-Out

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: MS-based comparative enrichment analysis of VGluT3 immunoisolates’ proteome reveals age-dependent changes in the expression of synaptic and trafficking proteins . A , schematic representation of the immunoisolation approach to isolate IHC VGluT3-positive vesicular structures from immature (P8) and mature (P23) organs of Corti. S2 fraction was prepared as described in Figure 1 from ∼100 organs of Corti (per biological replicate) and used as starting material in VGluT3-and control IgG-specific immunoisolations. Two technical replicates (indicated as T1 and T2) from two independent immunoisolation procedures (biological replicates R1 and R2) were measured. B and C , approach used for the analysis of the MS data. B , protein enrichment was assessed by comparing VGluT3 immunoisolates with both control IgG immunoisolates and input S2 samples. The –log 10 adjusted p value was plotted against the log 2 iBAQ fold change of VGluT3 over control (IgG or input), with a significant t test FDR threshold of 5% and S 0 = 0. C , to visualize protein enrichment in VGluT3 immunoisolates as compared to both control IgG and input S2, log 2 iBAQ fold change VGluT3/Control IgG vs. log 2 iBAQ fold change VGluT3/Input was plotted; proteins in the upper right quadrant were enriched in VGluT3 immunoisolates – a threshold of log 2 iBAQ fold difference >0.7 was set to consider only proteins with at least 1.5-fold enrichment. D and E , Scatter plots showing differential enrichment of proteins in VGluT3 immunoisolates when compared to both control IgG and inputs at P8 ( D ) and P23 ( E ); displayed are IHC marker proteins VGluT3 and otoferlin, classical SV proteins, SNAREs, SNARE-binding proteins and other proteins. Numbers in parenthesis refer to the total number of significantly enriched proteins in VGluT3 immunoisolates over control IgG and Input (>1.5-fold enrichment). Gene names are displayed. Stx12 gene annotated in UniProt refers to syntaxin-12/13 protein (syntaxin-12 and syntaxin-13 are the same protein; syntaxin-13 is the accepted term by the scientific community, but is still annotated in most databases as syntaxin-12). See also supplemental Fig. S3 for individual volcano plots. Source data are available for this figure ( supplemental Table S2 ). IHC, inner hair cell; MS, mass spectrometry; SV, synaptic vesicle.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Expressing, Protein Enrichment, Marker, Binding Assay, Mass Spectrometry

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Comparative analysis of enriched proteins in P8 and P23 VGluT3 immunoisolates revealed a small overlap in protein identifications between ages and a different relative enrichment for shared proteins . A , Venn diagram showing the overlap between the significantly enriched proteins (>1.5-fold enrichment) at P8 and P23. B and C , ranking of significantly enriched proteins according to their relative enrichment in VGluT3 over Control IgG immunoisolates and input S2, before ( B ) and after ( C ) hearing onset. Relative Enrichment corresponds to the average of log 2 iBAQ (VGluT3/Input) and log 2 iBAQ (VGluT3/Control IgG). Displayed are only enriched proteins shown also in Figure 4 , D and E ( upper right quadrant and >1.5-fold enrichment). Stx12 gene annotated in UniProt refers to syntaxin-12/13 protein (syntaxin-12 and syntaxin-13 are the same protein; syntaxin-13 is the accepted term by the scientific community, but is still annotated in most databases as syntaxin-12). Source data are available for this figure ( supplemental Table S3 ).

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Functional annotation of mature VGluT3-associated IHC proteome reveals a mixed SV-endosomal signature . A , sunburst diagram with functional annotation of the enriched proteins in VGluT3 immunoisolates at P23. Proteins were grouped for display according to their cellular component and their involvement in trafficking events. Annotations were done manually and based on information available in several databases; proteins were grouped according to cellular component and biological function. For detailed annotations see supplemental Table S4 . B–F , Scatter plots showing positively enriched proteins in VGluT3 immunoisolates at P23 when compared to both control IgG and input; log 2 iBAQ fold change VGluT3/Control IgG vs. log 2 iBAQ fold change VGluT3/Input was plotted. Displayed are proteins involved in trafficking events in different trafficking organelles (SV, endolysosomal, Golgi, and ER proteins) including SNAREs and resident proteins. Several Rab GTPase proteins, mostly of endolysosomal nature, were also enriched ( F ). Annotations were done manually and based on information available in several databases; proteins were grouped according to cellular compartment and biological function. For detailed annotation see supplemental Table S4 . In ( B – F ) gene names are displayed. Stx12 gene annotated in UniProt refers to syntaxin-12/13 protein (syntaxin-12 and syntaxin-13 are the same protein; syntaxin-13 is the accepted term by the scientific community but is still annotated in most databases as syntaxin-12). For ranking of proteins according to their relative enrichment, see supplemental Fig. S5 . Source data are available for this figure ( supplemental Table S2 ). ER, endoplasmic reticulum; PM, plasma membrane; SV, synaptic vesicle.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Functional Assay, Membrane

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals the Composition of Glutamatergic Organelles of Auditory Inner Hair Cells

doi: 10.1016/j.mcpro.2023.100704

Figure Lengend Snippet: Immunolocalization analysis of SNARE, SV, and kinase proteins in the adult organ of Corti . A–D , VAMP-7 ( A ), syntaxin-12/13 ( B ), syntaxin-8 ( C ), and syntaxin-7 ( D ), all highly enriched in our MS experiments, are expressed in IHCs. E–H , some classical neuronal SV proteins SCAMP1 ( E ), V-ATPase ( F ), and SV2B ( G ), and the kinase PKCα ( H ), all enriched in our MS experiments, are expressed in IHCs. Note that all proteins localize to the basolateral region of the IHCs, although in some cases the protein is also expressed in supporting cells and/or in afferent (postsynaptic) and efferent fibers of SGNs. Images correspond to high magnification views of representative P15–25 IHCs immunolabeled with antibodies against the candidate proteins ( red ), the ribbon marker CtBP2/RIBEYE ( green ), and the IHC marker otoferlin ( blue ). The upper panels show overviews of representative IHCs, displaying maximum intensity projections of 5 to 10 confocal optical sections through the longitudinal axis of the IHCs (scale bars: 5 μm). The bottom panels show a zoom into the synaptic area, displaying single confocal optical sections through the longitudinal axis of a single IHCs at the basal region (scale bars: 2 μm). IHC, inner hair cell; MS, mass spectrometry; SGN, spiral ganglion neuron.

Article Snippet: The following primary antibodies were used: rabbit anti-VAMP-7 (232003, Synaptic Systems), rabbit anti-syntaxin-6 (110062, Synaptic Systems), rabbit anti-syntaxin-7 (110072, Synaptic Systems), rabbit anti-syntaxin-8 (110083, Synaptic Systems), rabbit anti-syntaxin-12/13 (110133, Synaptic Systems), rabbit anti-syntaxin-16 (110162, Synaptic Systems), rabbit anti-SCAMP1 (PA1-739, Thermo Fisher Scientific), mouse anti-V-ATPase (149011, Synaptic Systems), mouse anti-SV2B (119111, Synaptic Systems), rabbit anti-Vti1A (165002, Synaptic Systems), rabbit anti-PKC alpha [Y124] (ab32376, Abcam), rabbit anti-YKT6 (ab236583, Abcam), rabbit anti-VAP-A (249002, Synaptic Systems), mouse anti-VCP (MA3-004, Thermo Fisher Scientific), mouse anti-otoferlin [13A9] (ab53233, Abcam), rabbit anti-otoferlin (178003, Synaptic Systems), rabbit anti-VGluT3 (135203, Synaptic Systems), guinea pig anti-VGluT3 (135204, Synaptic Systems), mouse anti-synapsin-1 (106001, Synaptic Systems), goat IgG anti-CtBP2 [E−16] (sc-5967, Santa Cruz Biotechnology), rabbit anti-Munc18-1 (116002, Synaptic Systems), rabbit anti-Munc18-2 (116102, Synaptic Systems), rabbit anti-Munc18-3 (116202, Synaptic Systems), and mouse anti-VAMP-2 (104211, Synaptic Systems).

Techniques: Immunolabeling, Marker, Mass Spectrometry