rabbit anti-rigi  (Proteintech)

Journal: Autophagy

Article Title: Picornavirus VP2 protein suppresses innate immunity through selective autophagic degradation of IKBKE/IKKε

doi: 10.1080/15548627.2025.2597460

Figure Lengend Snippet: SVA VP2 protein inhibits the expression of IKBKE and interacts with it. (A) HEK-293T cells were transfected with empty vector or Flag-VP2 plasmids. The cells were lysed at 24 hpt and analyzed by western blotting using the indicated antibodies. (B-D) HEK-293T cells were transfected with 0, 0.25, 0.5 or 1 μg of Flag-VP2 expressing plasmids for 24 h. The expression levels of endogenous IKBKE (B), RIGI (C), or IRF3 (D) were assessed by western blotting. (E) HEK-293T cells were transfected with Flag-VP2 expressing plasmids (0, 0.25, 0.5 or 1 μg) for 24 h. Total rna was extracted from the cells, and IKBKE mRNA levels were quantified by qPCR. (F, G) HEK-293T cells were infected with SVA (MOI = 0.5) for 0, 4, 8 and 12 h respectively, and the protein expression of IKBKE was analyzed by western blotting (F), the mRNA expression of IKBKE was detected by qPCR (G). (H) HEK-293T cells were co-transfected with Flag-VP2 and either Vec, various HA-tagged innate immune molecule-expressing plasmids (MDA5, RIGI, MAVS, TRAF6, TBK1, IRF3, IRF7, IKBKE, and tank) or MYC-tagged TRAF3. At 36 hpt, the cell lysates were subjected to co-IP assay analysis. The immunoprecipitated proteins and whole-cell lysates (WCL) were analyzed by western blotting using the specified antibodies. (I) HEK-293T cells were co-transfected with HA-IKBKE and vector or Flag-VP2 expressing plasmids for 36 h. The cell lysates were immunoprecipitated with anti-HA or control IgG antibodies, and the antigen-antibody complex was subjected to western blotting analysis. (J) HEK-293T cells were mock-infected or infected with SVA at an MOI of 0.5 for 12 h. Cell lysates were then immunoprecipitated with anti-VP2 or control IgG antibodies, the antigen-antibody complex were assessed by western blotting. (K) HEK-293T cells were mock-infected or infected with SVA (MOI = 0.1) for 10 h, after which the colocalization of IKBKE (red) and VP2 (green) was assessed by immunofluorescence assay (IFA). Nuclei were counterstained with DAPI (blue). (l, M) HEK-293T cells were co-transfected with increasing amounts of Flag-VP2 and MYC-IKBKE, along with HA-TBK1 (L) or HA-IRF3 (M) expressing plasmids. At 24 hpt, the cells were treated with SeV for another 12 h. The cells were lysed and immunoprecipitated with anti-MYC antibodies. The immunoprecipitated proteins and WCL were analyzed by western blotting using the specified antibodies.

Article Snippet: The commercial antibodies used in this study include: anti-Flag mouse Ab (Sigma, F1804), anti-MYC mouse Ab (Sigma, M5546), anti-HA mouse Ab (Proteintech, 66,006–2-Ig), anti-RIGI rabbit Ab (Cell Signaling Technology, 3743S), anti-IFIH1/MDA5 rabbit Ab (Abcam, Ab126630 ), anti-MAVS rabbit Ab (Cell Signaling Technology, 3993S), anti-TRAF3 rabbit Ab (Cell Signaling Technology, 4729T), anti-TRAF6 rabbit Ab (Cell Signaling Technology, 67591S), anti-TBK1 rabbit Ab (Cell Signaling Technology, 38066S), anti-IRF3 rabbit Ab (Proteintech, 11,312–1-AP), anti-IRF7 rabbit Ab (Abcam, ab109255), anti-LC3 rabbit Ab (Proteintech, 14,600–1-AP), anti-TUBB/β-tubulin mouse Ab (Abclonal, A12289), anti-IKBKE/IKKε rabbit Ab (Abclonal, A3463), anti-K33 rabbit Ab (Abclonal, A18199), anti-RNF114 rabbit Ab (Abclonal, A10636), anti-CALCOCO2/NDP52 rabbit Ab (Abclonal, A24021), anti-SQSTM1/p62 rabbit Ab (Abclonal, A19700).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Infection, Co-Immunoprecipitation Assay, Immunoprecipitation, Control, Immunofluorescence

Journal: JCI insight

Article Title: DNA hypomethylation promotes transposable element expression and activation of immune signaling in renal cell cancer.

doi: 10.1172/jci.insight.137569

Figure Lengend Snippet: Figure 3. Decitabine treatment activates antiviral signaling in ccRCC cells. 786-0 cells were cultured for 3 days with decitabine, cells were harvested on day 5 and assayed by RNAseq. RNAseq was performed in duplicate samples (n = 2) for each experimental condition. (A) Lollipop representation of signifi- cantly enriched pathways for genes upregulated by decitabine treatment (300 nM) in 786-0 cells as compared with DMSO vehicle (RNA-Seq; Wald test, FDR < 0.05 and log2-fold > 1.5). Lollipop height and intensity color scale indicate magnitude –log10 (FDR) of the enrichment for each pathway. The number of genes enriched in that pathway is indicated inside each lollipop gene enriched in that pathway. (B) Expression levels of antiviral response genes in 786-0 cells treated with 0 nM, 100 nM, and 300 nM decitabine. Heatmap rows show mean expression of duplicate RNAseq measurements. Significance (100 nM versus 0 nM; 300 nM versus 0 nM) assessed by Wald test and corrected for multiple testing (FDR). *FDR < 0.05 and**FDR < 0.001. (C) Antiviral protein levels were assessed by immunoblot analysis. 786-0 cells were treated with 100 nM decitabine for 3 days (days 0, 1, and 2), and protein was harvested at days 1–5. Mock-treated cells were cultured with DMSO for 3 days, and protein was harvested at day 5. Protein expression levels of DNMT1, RIGI, MDA5, IRF7, ISG15, and β-actin were assessed by immunoblot. β-Actin was used as a loading control.

Article Snippet: Antibodies used included rabbit anti-DNMT1 (1:1000; clone D63A6 XP mAb, Cell Signaling Technology, 5032), -IRF7 (1:1000; Cell Signaling Technology, 4920), -RIGI (1:500; clone D33H10, Cell Signaling Technology, 4200), -MDA5 (1:500; clone D10F10, Cell Signaling Technology, 5321) and –β-actin (1:10000; Abcam, ab8227), and mouse anti– ISG15 mAb (1:500; clone F-9, Santa Cruz Biotechnology sc-166755).

Techniques: Cell Culture, RNA Sequencing, Expressing, Western Blot, Control

Journal: JCI insight

Article Title: DNA hypomethylation promotes transposable element expression and activation of immune signaling in renal cell cancer.

doi: 10.1172/jci.insight.137569

Figure Lengend Snippet: Figure 4. KO of MDA5, RIGI, or MAVS attenuates antiviral signaling in ccRCC cells. (A) Verification of MAVS-, MDA5-, and RIGI-KO cells in indicated 786-0 cell lines by immunoblot analysis. β-Actin was used as a loading control. (B–E) Antiviral signaling is attenuated by MDA5-, RIGI-, or MAVS-KO 786-0 cells, but not in scrambled control cells. Antiviral signaling was assessed by qPCR of IRF7, ISG15, MX1, and OAS3 genes. Black dot and line indicate mean ± SD (n = 3). Significance (MAVS/RIGI/MDA5-KO [100 nM] versus Scramble [100 nM]) assessed by 2-tailed t test, and P values were adjusted via Holm-Bonferroni correction. *P < 0.05. (F and G) ERV expression is activated by decitabine treatment of in MAVS-, MDA5-, and RIGI-KO in 786-0 cell lines and scrambled controls. ERV3-2 and ERV4700 expression measured by qPCR.

Article Snippet: Antibodies used included rabbit anti-DNMT1 (1:1000; clone D63A6 XP mAb, Cell Signaling Technology, 5032), -IRF7 (1:1000; Cell Signaling Technology, 4920), -RIGI (1:500; clone D33H10, Cell Signaling Technology, 4200), -MDA5 (1:500; clone D10F10, Cell Signaling Technology, 5321) and –β-actin (1:10000; Abcam, ab8227), and mouse anti– ISG15 mAb (1:500; clone F-9, Santa Cruz Biotechnology sc-166755).

Techniques: Western Blot, Control, Expressing

Journal: JCI insight

Article Title: DNA hypomethylation promotes transposable element expression and activation of immune signaling in renal cell cancer.

doi: 10.1172/jci.insight.137569

Figure Lengend Snippet: Figure 5. MDA5 and RIGI bind ERVs induced by DNA hypomethylation. (A) Verification of Doxyline (DOX) inducible MDA5-FLAG and RIGI-FLAG proteins in 786-0 cell lines by immunoblot analysis. Indicated cell lines were treated with and without DOX and decitabine. Addition of DOX induces expression of MDA5-FLAG or RIGI-FLAG constructs. MDA5- FLAG and RIGI-FLAG protein was detected using anti–FLAG M2 antibody. β-Actin was used as a loading control. (B–E) Enrichment of ERVs (ERVE, ERVFC1, ERVFC2, ERV3-2, and ERV4700) bound by MDA5 (B and C) and RIGI (D and E) was assessed by RIP. IP of MDA5-FLAG and RIGI-FLAG was performed using anti–FLAG M2 antibody. ERV expression was assessed by qPCR. Values shown as fold-enrichment over input of the 100 nM decitabine-treatment group, normalized to the 0 nM decitabine-treatment group. Black dot and line indicate mean ± SD (n = 3). Significance (100 nM fold-en- richment MDA5/RIGI-FLAG IP versus 0 nM fold-enrichment MDA5/RIGI-FLAG IP) assessed by 2-tailed t test, and P values were adjusted via Holm-Bonferroni correction. *P < 0.05.

Article Snippet: Antibodies used included rabbit anti-DNMT1 (1:1000; clone D63A6 XP mAb, Cell Signaling Technology, 5032), -IRF7 (1:1000; Cell Signaling Technology, 4920), -RIGI (1:500; clone D33H10, Cell Signaling Technology, 4200), -MDA5 (1:500; clone D10F10, Cell Signaling Technology, 5321) and –β-actin (1:10000; Abcam, ab8227), and mouse anti– ISG15 mAb (1:500; clone F-9, Santa Cruz Biotechnology sc-166755).

Techniques: Western Blot, Expressing, Construct, Control