anti irf3 rabbit polyclonal antibody  (Santa Cruz Biotechnology)

Journal: The Journal of Clinical Investigation

Article Title: An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP

doi: 10.1172/JCI144339

Figure Lengend Snippet: ( A ) The effect of cGAMP on the viability of B16 cells cocultured with or without splenocytes from WT or STING-deficient mice ( Tmem173 gt ) was assessed by MTT assay ( n = 3). ** P < 0.01, by 2-tailed, paired Student’s t test. ( B ) Expression of CD69 in WT or Tmem173 gt splenocytes treated with vehicle or cGAMP was detected by flow cytometry. ( C ) Expression of CD69 in WT or Tmem173 gt NK cells treated with vehicle or cGAMP was detected by flow cytometry. ( D ) Expression of CD86 in WT or Tmem173 gt myeloid cells treated with vehicle or cGAMP was detected by flow cytometry. ( E ) Expression of p-TBK1 and p-IRF3 was detected by Western blotting in WT or Tmem173 gt splenocytes treated with vehicle, cGAMP, or DMXAA, respectively. ( F ) Production of IFN-β in WT or Tmem173 gt splenocytes treated with vehicle or cGAMP was detected by ELISA ( n = 3). ** P < 0.01, by 2-tailed, paired Student’s t test. ( G ) Production of IFN-β in WT splenocytes treated with different concentrations of cGAMP, c-di-GMP, or DMXAA was detected by ELISA ( n = 3). * P < 0.05, by 2-way ANOVA. Data are presented as the mean ± SD.

Article Snippet: After blocking with 5% BSA at room temperature for 1 hour, membranes were incubated with the following primary antibodies: anti–STING rabbit polyclonal antibody (19851-1-AP, Proteintech) diluted at 1:600, anti–mouse TBK1 antibody (ab40676, Abcam) diluted at 1:1000; anti–mouse phosphorylated TBK1 (p-TBK1) (Ser172) antibody (ab109272, Abcam) diluted at 1:500; anti–mouse IRF3 rabbit polyclonal antibody (11312-1-AP, Proteintech) diluted at 1:1000; anti–mouse p–IRF-3 (Ser396) rabbit mAb (29047, Cell Signaling Technology) diluted at 1:1000; anti–mouse cGAS antibody (ab252416, Abcam) diluted at 1:1000; anti–mouse SLC19A1 antibody (25958-1-AP, Proteintech) diluted at 1:1000; and anti–GAPDH mouse mAb (60004-1-Ig, Proteintech) diluted at 1:1000.

Techniques: MTT Assay, Expressing, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP

doi: 10.1172/JCI144339

Figure Lengend Snippet: ( A ) B16 Tmem173–/– cells stably expressing SEAP to monitor IFN activity were transfected with erSTING-Flag, pmSTING-Flag, or mutated pmSTING-Flag (Mut pmSTING-Flag) at the TBK1 phosphorylation site (Ser316R). SEAP was detected in these cells after treatment with vehicle or cGAMP ( n = 3). * P < 0.05, by 2-tailed, paired Student’s t test. ( B ) Western blotting was performed to detect p-TBK1 and p-IRF3 in B16 Tmem173–/– cells transfected with erSTING-Flag or pmSTING-Flag upon treatment with vehicle or cGAMP. ( C and D ) B16 Tmem173–/– cells were transfected with both erSTING-Flag and erSTING-EGFP or both pmSTING-Flag and pmSTING-EGFP, and then treated with vehicle or cGAMP. Immunoprecipitation using anti-Flag antibody ( C ) or anti-GFP antibody ( D ) was performed to detect pmSTING or erSTING dimerization in response to extracellular cGAMP. ( E ) B16 Tmem173–/– cells were transfected with erSTING-Flag or pmSTING-Flag and then treated with vehicle or cGAMP. Immunoprecipitation using anti-Flag antibody was performed to detect the interaction between pmSTING (or erSTING) and TBK1 or IRF3, respectively.

Article Snippet: After blocking with 5% BSA at room temperature for 1 hour, membranes were incubated with the following primary antibodies: anti–STING rabbit polyclonal antibody (19851-1-AP, Proteintech) diluted at 1:600, anti–mouse TBK1 antibody (ab40676, Abcam) diluted at 1:1000; anti–mouse phosphorylated TBK1 (p-TBK1) (Ser172) antibody (ab109272, Abcam) diluted at 1:500; anti–mouse IRF3 rabbit polyclonal antibody (11312-1-AP, Proteintech) diluted at 1:1000; anti–mouse p–IRF-3 (Ser396) rabbit mAb (29047, Cell Signaling Technology) diluted at 1:1000; anti–mouse cGAS antibody (ab252416, Abcam) diluted at 1:1000; anti–mouse SLC19A1 antibody (25958-1-AP, Proteintech) diluted at 1:1000; and anti–GAPDH mouse mAb (60004-1-Ig, Proteintech) diluted at 1:1000.

Techniques: Stable Transfection, Expressing, Activity Assay, Transfection, Phospho-proteomics, Western Blot, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP

doi: 10.1172/JCI144339

Figure Lengend Snippet: ( A ) 293T cells were cotransfected with h-erSTING-Flag or h-pmSTING-Flag and a luciferase reporter to detect IFN production. Luciferase activity was detected in these cells after treatment with vehicle or cGAMP ( n = 3). * P < 0.05, by 2-tailed, paired Student’s t test. ( B ) Western blotting was performed to detect p-TBK1 and p-IRF3 levels in 293T cells transfected with h-erSTING-Flag or h-pmSTING-Flag upon treatment with vehicle or cGAMP. ( C and D ) 293T cells were transfected with both h-erSTING-Flag and h-erSTING-EGFP or both h-pmSTING-Flag and h-pmSTING-EGFP, and then treated with vehicle or cGAMP. Immunoprecipitation using anti-Flag antibody ( C ) or anti-GFP antibody ( D ) was performed to detect the dimerization of h-pmSTING or h-erSTING in response to extracellular cGAMP. ( E ) 293T cells were transfected with h-erST1 ING-Flag or h-pmSTING-Flag and then treated with vehicle or cGAMP. Immunoprecipitation using anti-Flag antibody was performed to detect the interaction between h-pmSTING (or h-erSTING) and TBK1 or IRF3, respectively.

Article Snippet: After blocking with 5% BSA at room temperature for 1 hour, membranes were incubated with the following primary antibodies: anti–STING rabbit polyclonal antibody (19851-1-AP, Proteintech) diluted at 1:600, anti–mouse TBK1 antibody (ab40676, Abcam) diluted at 1:1000; anti–mouse phosphorylated TBK1 (p-TBK1) (Ser172) antibody (ab109272, Abcam) diluted at 1:500; anti–mouse IRF3 rabbit polyclonal antibody (11312-1-AP, Proteintech) diluted at 1:1000; anti–mouse p–IRF-3 (Ser396) rabbit mAb (29047, Cell Signaling Technology) diluted at 1:1000; anti–mouse cGAS antibody (ab252416, Abcam) diluted at 1:1000; anti–mouse SLC19A1 antibody (25958-1-AP, Proteintech) diluted at 1:1000; and anti–GAPDH mouse mAb (60004-1-Ig, Proteintech) diluted at 1:1000.

Techniques: Luciferase, Activity Assay, Western Blot, Transfection, Immunoprecipitation

Journal: The Journal of Clinical Investigation

Article Title: An alternatively spliced STING isoform localizes in the cytoplasmic membrane and directly senses extracellular cGAMP

doi: 10.1172/JCI144339

Figure Lengend Snippet: ( A ) Multiple protein sequence alignment of STING proteins orthologous to the mouse pmSTING isoform. The STING proteins in different species shown here meet the following criteria: (a) possess an odd number of predicted TM domains; and (b) possess a C-terminal domain identical to that of the respective species’ canonical STING protein. ( B ) Model of the generation of 2 alternatively spliced STING isoforms and how they sense extracellular and intracellular cGAMP, respectively. Two STING transcripts generated by alternative splicing are translated in the cytoplasm and transported to the pmSTING and the endoplasmic reticulum (erSTING), respectively. Upon binding the intracellular cGAMP synthesized by cytosolic DNA–activated cGAS, erSTING undergoes homodimerization and translocates from endoplasmic reticulum to the perinuclear area, where it recruits and activates TBK1, which phosphorylates the transcription factor IRF3 and results in the translocation of IRF3 from the cytoplasm to the nucleus to induce the transcription of IFN and other immune cytokines. By contrast, the extracellular cGAMP released by dead cells directly binds pmSTING and causes homodimerization and translocation of pmSTING from the plasmatic membrane to the perinuclear area, where it activates TBK1/IRF3/IFN signaling.

Article Snippet: After blocking with 5% BSA at room temperature for 1 hour, membranes were incubated with the following primary antibodies: anti–STING rabbit polyclonal antibody (19851-1-AP, Proteintech) diluted at 1:600, anti–mouse TBK1 antibody (ab40676, Abcam) diluted at 1:1000; anti–mouse phosphorylated TBK1 (p-TBK1) (Ser172) antibody (ab109272, Abcam) diluted at 1:500; anti–mouse IRF3 rabbit polyclonal antibody (11312-1-AP, Proteintech) diluted at 1:1000; anti–mouse p–IRF-3 (Ser396) rabbit mAb (29047, Cell Signaling Technology) diluted at 1:1000; anti–mouse cGAS antibody (ab252416, Abcam) diluted at 1:1000; anti–mouse SLC19A1 antibody (25958-1-AP, Proteintech) diluted at 1:1000; and anti–GAPDH mouse mAb (60004-1-Ig, Proteintech) diluted at 1:1000.

Techniques: Sequencing, Generated, Alternative Splicing, Binding Assay, Synthesized, Translocation Assay, Membrane