Journal: PLoS Pathogens

Article Title: USP49 negatively regulates cellular antiviral responses via deconjugating K63-linked ubiquitination of MITA

doi: 10.1371/journal.ppat.1007680

Figure Lengend Snippet: (A) Immunoprecipitation (with anti-MITA) and immunoblot analysis (with anti-MITA or anti-USP49) of THP-1 or U937 cells infected with HSV-1, or transfected with ISD (10 μg) or cGAMP (4 μg) for the indicated time points. (B) Immunoprecipitation (with anti-Flag) and immunoblot analysis (with anti-FLAG or anti-HA) of HEK293 cells transfected with plasmids encoding HA-MITA and FLAG-tagged USP49 or USP49 truncates for 24 h or transfected with plasmids encoding FLAG-USP49 and HA-MITA or MITA truncates for 24 h. (C) qRT-PCR analysis of IFNB , IFNA4 , CCL5 and IL6 in control and USP49 KO THP-1 cells infected with HSV-1 or SeV for 0–6 h. (D) Immunoblot analysis of phosphorylated and total IRF3, IκBα, TBK1 and Tubulin in control and USP49 KO THP-1 cells infected with HSV-1 or SeV for 0–6 h. * P < 0.05; ** P < 0.01; *** P <0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments (mean ± S.D. in C).

Article Snippet: Mouse control IgG (Santa Cruz Biotechnology, sc-2025) and rabbit control IgG (Millipore, 12–370), HRP-conjugated goat-anti mouse or rabbit IgG (Thermo Scientific, PA1-86717 and SA1-9510) (1:3000), HRP-conjugated mouse anti-FLAG (Sigma, A8592)(1:1000), mouse anti-FLAG (Sungene, KM8002)(1:2000), anti-GFP (Sungene, KM8009)(1:2000) anti-β-Actin (KM9001)(1:2000), anti-Tubulin (KM9003), anti-GAPDH(KM9002), anti-HA (COVANCE, MMS-101R)(1:2000), anti-Ubiquitin (sc-8017)(1:500), anti-ubiquitin K63-specific linkage (Millipore 05–1308)(1:500), rabbit anti-TBK1(Abcam, 96328–11), anti-p-TBK1(Abcam, 109272), anti-IRF3 (sc-9082)(1:1000), anti-p-IRF3 (Cell Singling Technologies, 4947S)(1:1000), anti-IκBα (sc-371)(1:1000), anti-p-IκBα (Cell Singling Technologies, 9246L)(1:1000), anti-USP49 (proteintech,18066-1-AP), anti-mouse MITA and anti-human MITA (Cell Singling Technologies,13647) (proteintech, 19851-1-AP ) were purchased from the indicated manufactures.

Techniques: Immunoprecipitation, Western Blot, Infection, Transfection, Quantitative RT-PCR, Control

Journal: PLoS Pathogens

Article Title: USP49 negatively regulates cellular antiviral responses via deconjugating K63-linked ubiquitination of MITA

doi: 10.1371/journal.ppat.1007680

Figure Lengend Snippet: (A) Denature-immunoprecipitation (Denature-IP) (with anti-FLAG or IgG as a control) and immunoblot analysis (with anti-FLAG, anti-HA, anti-K63-linked ubiquitin or anti-GFP) of HEK293 cells transfected with plasmids encoding FLAG-MITA, HA-Ubiquitin and empty vector or GFP-USP49 or GFP-USP49 (CA) for 24 h. (B) Pulldown (with GST beads and GST-TUBEs) and immunoblot analysis (with anti-FLAG, anti-HA or anti-GFP) of HEK293 cells transfected with plasmids encoding FLAG-MITA, HA-Ubiquitin and empty vector or GFP-USP49 or GFP-USP49 (CA) for 24 h. (C) Denature-IP (with anti-MITA) and immunoblot analysis (anti-K63-linked ubiquitin, anti-MITA or anti-β-Actin) of Usp49 +/+ and Usp49 -/- MLFs infected with HSV-1 for 0–3 h. (D) Pulldown (with GST beads and TUBE) and immunoblot analysis (anti-K63-linked ubiquitin, anti-MITA or anti-β-Actin) of Usp49 +/+ and Usp49 -/- MLFs infected with HSV-1 for 0–3 h. (E) Denature-IP (with anti-MITA) and immunoblot analysis (with anti-K63-linked Ub, anti-MITA, anti-FLAG or anti- anti-β-Actin) of Usp49 -/- MLFs reconstituted with empty vector, USP49 or USP49 (CA) infected with HSV-1 for 0–3 h. (F) Native–PAGE analysis and SDS–PAGE of the aggregation of MITA in Usp49 +/+ and Usp49 -/- MLFs or Usp49 -/- MLFs reconstituted with empty vector, USP49 or USP49 (CA) infected with HSV-1 for 0–4 h. (G) Immunoprecipitation (with anti-MITA) and immunoblot analysis (with anti-TBK1, anti-MITA, anti-USP49 or anti-β-Actin) MLFs infected with HSV-1 for 0–4 h. Data are representative of at least three independent experiments.

Article Snippet: Mouse control IgG (Santa Cruz Biotechnology, sc-2025) and rabbit control IgG (Millipore, 12–370), HRP-conjugated goat-anti mouse or rabbit IgG (Thermo Scientific, PA1-86717 and SA1-9510) (1:3000), HRP-conjugated mouse anti-FLAG (Sigma, A8592)(1:1000), mouse anti-FLAG (Sungene, KM8002)(1:2000), anti-GFP (Sungene, KM8009)(1:2000) anti-β-Actin (KM9001)(1:2000), anti-Tubulin (KM9003), anti-GAPDH(KM9002), anti-HA (COVANCE, MMS-101R)(1:2000), anti-Ubiquitin (sc-8017)(1:500), anti-ubiquitin K63-specific linkage (Millipore 05–1308)(1:500), rabbit anti-TBK1(Abcam, 96328–11), anti-p-TBK1(Abcam, 109272), anti-IRF3 (sc-9082)(1:1000), anti-p-IRF3 (Cell Singling Technologies, 4947S)(1:1000), anti-IκBα (sc-371)(1:1000), anti-p-IκBα (Cell Singling Technologies, 9246L)(1:1000), anti-USP49 (proteintech,18066-1-AP), anti-mouse MITA and anti-human MITA (Cell Singling Technologies,13647) (proteintech, 19851-1-AP ) were purchased from the indicated manufactures.

Techniques: Immunoprecipitation, Control, Western Blot, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Infection, Clear Native PAGE, SDS Page

Journal: PLoS Pathogens

Article Title: USP49 negatively regulates cellular antiviral responses via deconjugating K63-linked ubiquitination of MITA

doi: 10.1371/journal.ppat.1007680

Figure Lengend Snippet: HSV-1 infection induces production of cGAMP that binds to MITA and leads to K63-linked ubiquitination and oligomerization of MITA. USP49 removes K63-linked ubiquitin chains from MITA and thereby inhibits the oligomerization. Such a deubiquitination impairs the subsequent recruitment of TBK1 and phosphorylation of IRF3 after HSV-1 infection.

Article Snippet: Mouse control IgG (Santa Cruz Biotechnology, sc-2025) and rabbit control IgG (Millipore, 12–370), HRP-conjugated goat-anti mouse or rabbit IgG (Thermo Scientific, PA1-86717 and SA1-9510) (1:3000), HRP-conjugated mouse anti-FLAG (Sigma, A8592)(1:1000), mouse anti-FLAG (Sungene, KM8002)(1:2000), anti-GFP (Sungene, KM8009)(1:2000) anti-β-Actin (KM9001)(1:2000), anti-Tubulin (KM9003), anti-GAPDH(KM9002), anti-HA (COVANCE, MMS-101R)(1:2000), anti-Ubiquitin (sc-8017)(1:500), anti-ubiquitin K63-specific linkage (Millipore 05–1308)(1:500), rabbit anti-TBK1(Abcam, 96328–11), anti-p-TBK1(Abcam, 109272), anti-IRF3 (sc-9082)(1:1000), anti-p-IRF3 (Cell Singling Technologies, 4947S)(1:1000), anti-IκBα (sc-371)(1:1000), anti-p-IκBα (Cell Singling Technologies, 9246L)(1:1000), anti-USP49 (proteintech,18066-1-AP), anti-mouse MITA and anti-human MITA (Cell Singling Technologies,13647) (proteintech, 19851-1-AP ) were purchased from the indicated manufactures.

Techniques: Infection, Ubiquitin Proteomics, Phospho-proteomics

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: Mutation of K150 impairs the activity of MITA N153S . (A) RT-qPCR analysis of Ifnb , Isg15 , Isg56 , and Cxcl1 0 mRNA (left graphs) and immunoblot analysis of MITA, MITA K150N , MITA N153S , or MITA K150N/N153S (right panels) of Mita −/− MLFs reconstituted with wild-type (WT) or different MITA mutants. (B) ELISA analysis of CXCL1, TNF, and IL-6 in the supernatants of Mita −/− MLFs reconstituted with wild-type or different MITA mutants. ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.000 1; ns, not significant (one-way ANOVA). The graphs show mean ± SD. The data are representative of three independent experiments.

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Mutagenesis, Activity Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: The SAVI phenotypes are abolished in MITA K150N/N153S mice. (A) Representative images of 5-week-old WT, MITA N153S/WT and MITA NS/NS mice. (B) Survival analysis (Kaplan–Meier curves) of MITA N153S/WT ( n = 33) and MITA NS/NS ( n = 40) mice. (C) Spleen weight/body weight ratios (left) and representative images (right) of the spleens of 5-week-old WT ( n = 9), MITA N153S/WT ( n = 11), and MITA NS/NS ( n = 5) mice. (D) Representative images of HE-stained heart, kidney, and lung sections from 5-week-old WT ( n = 3), MITA N153S/WT ( n = 3), and MITA NS/NS ( n = 3) mice. (E) ELISA analysis of CXCL1, TNF, IL12p70, and IL-6 in the sera of 5-week-old WT ( n = 9), MITA N153S/WT ( n = 12) and MITA NS/NS ( n = 9) mice. (F–G) Flow cytometric analysis of splenocytes from 5-week-old WT ( n = 8), MITA N153S/WT ( n = 8), and MITA NS/NS ( n = 6) mice stained with fluorophore-conjugated antibodies against the indicated surface or intracellular molecules. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.000 1; ns, not significant (one-way ANOVA or the Log-Rank test). Scale bars represent 1 mm (black), 100 μm (purple), or 3 mm (red) in (D). The graphs show mean ± SD. The data are combined results from two independent experiments (B) or representative of three independent experiments (A, C-G).

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: MITA NS binds and responds to cGAMP. (A) Immunofluorescence staining of Calnexin (an ER marker) (red), GM130 (a Golgi apparatus marker) (red), and FLAG (green) and confocal microscopy analysis of Mita −/− MLFs reconstituted with FLAG-tagged MITA, MITA K150N , MITA N153S or MITA NS and treated with or without cGAMP (1 μg/mL) for 4 h, and co-localization analysis was conducted. (B) RT-qPCR analysis of Ifnb , Il6 , Isg56 , and Cxcl1 0 mRNA (left graphs) and immunoblot analysis of MITA, MITA K150N , MITA N153S , or MITA NS of Mita −/− MLFs reconstituted with FLAG-tagged MITA WT , MITA K150N , MITA N153S , or MITA NS (right panels) followed by treatment with cGAMP (1 μg/mL) for 0–4 h. (C) cGAMP binding assays of MITA WT , MITA K150N , MITA N153S , or MITA NS that were transfected into HEK293 cells followed by in vitro pulldown by Biotin-cGAMP. ∗ P < 0.05, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.000 1; ns, not significant (one-way ANOVA). Scale bars represent 10 μm (white) or 1 μm (cyan). The graphs show mean ± SD. The data are representative of two independent experiments.

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Immunofluorescence, Staining, Marker, Confocal Microscopy, Quantitative RT-PCR, Western Blot, Binding Assay, Transfection, In Vitro

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: Structural analyses of apo-MITA NS and cGAMP/MITA NS complex. (A) Cryo-EM map of apo-MITA NS (left), and cartoon representation showing domain-swapped dimer conformation with crossed hinge region (middle), and the hinge region (right) of the MITA NS dimer. (B) Structural comparison between wild-type apo-MITA (PDB: 6NT5 ) and apo-MITA NS (PDB: 9VXT ). The shift of connector loop in apo-MITA NS relative to it in wild-type apo-MITA is indicated by red arrow. Inset: the residues located around hinge region were shown (middle panel: apo-MITA NS ; right panel: wild-type apo-MITA). (C) Cryo-EM map of cGAMP/MITA NS filament (tetramer shown) (upper left) (PDB ID: 9VXU ), cartoon presentation of cGAMP/MITA NS filament (tetramer shown) (upper right), and interactions between LBDs (lower left) and TM (lower middle) from adjacent cGAMP/MITA NS dimer and interaction network in the hinge region of cGAMP/MITA NS dimer (lower right). The scheme on the right shows the connector region in a parallel conformation. (D) Structural comparison between cGAMP/MITA dimer (PDB: 8IK3 ) and cGAMP/MITA NS (PDB: 9VXU ). Inset: the residues located around the hinge region were shown (middle panel: cGAMP/MITA NS ; right panel: cGAMP/MITA). TM: transmembrane domain; LBD: ligand-binding domain.

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Cryo-EM Sample Prep, Comparison, Ligand Binding Assay

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: iRhom2 promotes SAVI progression in MITA N153S/WT mice. (A) Survival (Kaplan-Meier curves) of MITA N153S/WT →WT ( n = 25) and iRhom2 −/− MITA N153S/WT →WT ( n = 17) bone marrow chimeric mice. (B) Weight changes of MITA N153S/WT →WT ( n = 21) and iRhom2 −/− MITA N153S/WT →WT ( n = 17) chimeric mice. (C) Spleen weight/body weight ratios (upper) and representative images (lower) of spleens from MITA N153S/WT →WT ( n = 9) and iRhom2 −/− MITA N153S/WT →WT ( n = 8) chimeric mice at the 7th week after bone marrow transfer. (D) Representative images of HE-stained heart and lung sections of MITA N153S/WT →WT ( n = 3) and iRhom2 −/− MITA N153S/WT →WT ( n = 3) chimeric mice at the 7th week after bone marrow transfer. (E) ELISA of CXCL1 and TNF in the sera of MITA N153S/WT →WT ( n = 8) and iRhom2 −/− MITA N153S/WT →WT ( n = 8) chimeric mice at the 7th week after bone marrow transfer. (F–G) Flow cytometric analysis of splenocytes from MITA N153S/WT →WT ( n = 6) and iRhom2 −/− MITA N153S/WT →WT ( n = 8) chimeric mice at the 7th week after bone marrow transfer. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.000 1; ns, not significant (one-way ANOVA, two-tailed Student's t -test, or Log-Rank test). Scale bars represent 1 mm (black), 100 μm (purple), or 3 mm (red) in (D). The graphs show mean ± SD. The data are combined results from two independent experiments (A–B) or representative of two independent experiments (C–G).

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: iRhom2 is required for the Golgi localization of MITA GOFs. (A) Immunofluorescence staining of Calnexin (an ER marker) (red), GM130 (a Golgi apparatus marker) (red), and FLAG (green) and confocal microscopy analysis of iRhom2 +/+ or iRhom2 −/− MLFs reconstituted with FLAG-tagged MITA N153S , MITA V146L , or MITA V154M , and co-localization analysis was conducted. (B) Immunofluorescence staining of Calnexin (an ER marker) (red), GM130 (a Golgi apparatus marker) (red), and FLAG (green) and confocal microscopy analysis of iRhom2 +/+ or iRhom2 −/− MLFs reconstituted with FLAG-MITA NS followed by transfection with cGAMP (1 μg/mL) for 0–4 h, and co-localization analysis was conducted. Scale bars represent 10 μm (white) or 1 μm (cyan). The data are representative of two independent experiments.

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Immunofluorescence, Staining, Marker, Confocal Microscopy, Transfection

Journal: Cell Insight

Article Title: An additional site mutation in MITA/STING gain-of-function mutants abolishes the autoimmune SAVI phenotypes and directs a therapeutic strategy

doi: 10.1016/j.cellin.2026.100298

Figure Lengend Snippet: The SAVI inhibitory peptide abrogates the autoimmune SAVI phenotypes in MITA N153S/WT chimeric mice. (A) RT-qPCR analysis of Ifnb, Ifna4, and Il12p4 0 mRNA levels in MITA WT/WT or MITA N153S/WT MLFs treated with TAT-WT or TAT-SIP1/2 (0.5 μM, 2 μM or 5 μM) for 0–24 h. (B) Immunoprecipitation (with control IgG or an anti-MITA antibody) and immunoblot analysis (with anti-iRhom2 and anti-MITA antibodies) of MITA N153S/WT MLFs treated with TAT-WT or TAT-SIP1/2 (2 μM or 5 μM) for 0–24 h. (C) A scheme of TAT-SIP2 treatment (upper) and weight changes (lower) of MITA N153S/WT chimeric mice intraperitoneally injected with TAT-WT ( n = 9) or TAT-SIP2 ( n = 10) (20 mg/kg body weight) at the 5th week after bone marrow cell transfer for two weeks. (D) Representative images of spleens (upper) and spleen weight/body weight ratios (lower) of MITA N153S/WT chimeric mice intraperitoneally injected with TAT-WT ( n = 6) or TAT-SIP2 ( n = 7) every other day for two weeks starting from the 5th week after bone marrow cell transfer. (E) Survival (Kaplan–Meier curves) of MITA N153S/WT chimeric mice intraperitoneally injected with TAT-WT ( n = 18) or TAT-SIP2 ( n = 20) as described in (D). (F) Representative images of HE-stained heart, kidney, and lung sections from MITA N153S/WT chimeric mice treated with TAT-WT ( n = 3) or TAT-SIP2 ( n = 3) as described in (D). (G) ELISA of CXCL1, CCL5 and TNF in the sera of MITA N153S/WT chimeric mice treated with TAT-WT ( n = 6) or TAT-SIP2 ( n = 8) as described in (D). (H–I) Flow cytometric analysis of the splenocytes from MITA N153S/WT chimeric mice treated with TAT-WT ( n = 6) or TAT-SIP2 ( n = 6) as described in (D). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.000 1, ns, not significant; (one-way ANOVA, the two-tailed Student's t-test, or the Log-Rank test). Scale bars represent 1 mm (black), 100 μm (purple), or 3 mm (red) in (F). The graphs show mean ± SD. The data are combined results from two independent experiments (E) or representative of two independent experiments (A-C, D, F-I).

Article Snippet: The following antibodies and control IgG were purchased from the indicated manufacturers: mouse control IgG (Santa Cruz Biotechnology, sc-2025), mouse anti-GOLGA2/GM130 (Proteintech, 11308-1-AP), anti-RHBDF2 (iRhom2) (Abcepta, #AP13588A), anti-FLAG (Sigma, F3165), anti-HA (ABclonal, AE105), anti-Tubulin (ABclonal, A12289), and anti-STING (MITA) (Cell Signaling Technology, #13647 and ABclonal, A3575).

Techniques: Quantitative RT-PCR, Immunoprecipitation, Control, Western Blot, Injection, Staining, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: STING nuclear partners contribute to innate immune signaling responses

doi: 10.1016/j.isci.2021.103055

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-STING, D2P2F , Cell Signaling Technology , Cat# 13647; RRID: AB_2732796.

Techniques: Generated, Virus, Mutagenesis, Recombinant, Plasmid Preparation, Electron Microscopy, Transfection, Reverse Transcription, Luciferase, Control, Construct, Software

Journal: The Journal of Biological Chemistry

Article Title: Ring finger protein 5 activates sterol regulatory element–binding protein 2 (SREBP2) to promote cholesterol biosynthesis via inducing polyubiquitination of SREBP chaperone SCAP

doi: 10.1074/jbc.RA119.011849

Figure Lengend Snippet: RNF5 ubiquitinates lysine Lys-305 located in cytosolic loop 2 of SCAP. A, SRD-13A cells were seeded in 6-well plates and cultured until cell density reached 90% confluent. The cells were then switched to Opti-MEM and transfected with 0.5 μg/well HA-ubiquitin (Ub) and 1.0 μg/well WT or mutant Myc-SCAP in the presence or absence of 0.5 μg/well RNF5 expression plasmid and incubated for 2 h. The total amount of transfected plasmid was adjusted to 2 μg/well using pcDNA3.1 empty vector. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 20 h and then switched to fresh medium supplemented with 5% LPDS and 10 μm MG132 and cultured for another 2 h. The cells were then harvested for anti-Myc-SCAP IP. B, schematic presentation of the FLAG-SCAP plasmids used in (C). FL indicates full-length of SCAP, whereas plasmid no.1 to 4 are truncated from either C-terminal or N-terminal end of SCAP. TM indicates transmembrane domain, whereas L indicates loops pointed to the cytosol. Lysine residue Lys-305 on loop 2 is highlighted in red. C, SRD-13A cells were seeded as described in (A) and transfected with 2 μg/well empty vector or WT or truncated FLAG-SCAP plasmids in Opti-MEM and incubated for 2 h. The cells were then switched to DMEM/Ham's F-12 supplemented with 5% LPDS and cultured for 22 h. The cells were then harvested for anti-FLAG-SCAP IP. D, alignment of RNF5-binding region of SCAP and STING (14). Gray boxes indicate the amino acid sequences of transmembrane domains TM2 and TM3. White box indicates the amino acid sequence of cytosolic loop 2. The lysine residues that were ubiquitinated by RNF5 in SCAP and STING are highlighted in red.

Article Snippet: Anti-calnexin, anti-golgin-97, and anti-STING antibodies were purchased from Cell Signaling Technologies (codes: 2433, 13192, 13647).

Techniques: Cell Culture, Transfection, Ubiquitin Proteomics, Mutagenesis, Expressing, Plasmid Preparation, Incubation, Residue, Binding Assay, Sequencing