anti stat2 d9j7l  (Cell Signaling Technology Inc)

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Immunoblot analysis of STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells. The experiment was repeated twice. (B) Co-immunoprecipitation (coIP) analysis showing that STAT1 and STAT2 interact with PARP7. FLAG-PARP7 was ectopically expressed in HEK293T cells. FLAG-PARP7 was immunoprecipitated and then blotted for endogenous STAT1 and STAT2. The experiment was repeated twice. (C and D) Immunoblot analysis of ADP-ribosylated STAT1 (C) and STAT2 (D) in PARP7 WT and KO MEF cells. Cells were lysed in the presence of biotin-NAD + or NAD + . Proteins that were ADP-ribosylated by biotin-NAD + were pulled down with streptavidin beads and then blotted for STAT1 or STAT2. Samples treated with NAD + were used as a negative control. Both experiments were repeated three times. (E) PARP7 WT, but not the H532A mutant, ADP-ribosylates STAT1. GFP-tagged empty vector (EV), WT PARP7, or the H532 mutant were co-transfected with FLAG-STAT1 into HEK293T cells. FLAG-STAT1 was pulled down to detect interaction with PARP7 (blotted with GFP antibody) and ADP-ribosylation (blotted with ADP-ribose antibody). The experiment was repeated twice. See also .

Article Snippet: STAT1 (9172), phospho-STAT1 (Tyr701) (D4A7) (7649), STAT2 (D9J7L) (72604), phospho-Stat2 (Tyr690) (# 4441), ubiquitin (P4D1) (# 3936), TBK1/NAK (3013), phospho-TBK1/NAK (Ser172) (D52C2) (# 5483), SQSTM1/p62 (# 5114), Poly/Mono-ADP Ribose (D9P7Z) (# 89190), JAK1 (6G4) (# 3344), α-Tubulin (# 2144) antibodies were purchased from Cell Signaling.

Techniques: Western Blot, Immunoprecipitation, TNKS1 Histone Ribosylation Assay, Negative Control, Mutagenesis, Plasmid Preparation, Transfection

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT1 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm). Right: quantification of STAT1 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. (B) HEK293T cells were transfected with FLAG-WT or H532A mutant PARP7 overnight. FLAG-PARP7 was detected by immunofluorescence with anti-FLAG (green) antibodies. Endogenous STAT2 was detected by immunofluorescence with Alexa Flour 647. Nuclei were stained with Hoechst stain (blue) (scale bar: 2 μm.). Right: quantification of STAT2 and PARP7 co-localization using Pearson’s correlation coefficient. Data are presented as mean ± SD. *** p < 0.001, unpaired t test. All experiments were repeated twice. See also .

Article Snippet: STAT1 (9172), phospho-STAT1 (Tyr701) (D4A7) (7649), STAT2 (D9J7L) (72604), phospho-Stat2 (Tyr690) (# 4441), ubiquitin (P4D1) (# 3936), TBK1/NAK (3013), phospho-TBK1/NAK (Ser172) (D52C2) (# 5483), SQSTM1/p62 (# 5114), Poly/Mono-ADP Ribose (D9P7Z) (# 89190), JAK1 (6G4) (# 3344), α-Tubulin (# 2144) antibodies were purchased from Cell Signaling.

Techniques: Transfection, Mutagenesis, Immunofluorescence, Staining

Journal: Cell reports

Article Title: PARP7 inhibition stabilizes STAT1/STAT2 and relieves experimental autoimmune encephalomyelitis in mice

doi: 10.1016/j.celrep.2025.116130

Figure Lengend Snippet: (A) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after treatment of MG132 at 20 μМ (left) and cycloheximide (CHX) at 50 μМ (right). The experiment was repeated three times. (B) Western blot analysis of STAT1 and STAT2 in PARP7 WT or KO MEF cells after bafilomycin A1 treatment at 5 μМ. The experiment was repeated three times. (C) coIP analysis of p62 and PARP7 WT or H532A catalytic mutant. FLAG-tagged PARP7 WT and H532A mutant were transfected into HEK293T cells, and coIP was performed to detect interactions between p62 and PARP7 WT or H532A mutant. The experiment was repeated three times. (D) Co-localization of hemagglutinin (HA)-tagged p62 with GFP-tagged PARP7 WT or H532A mutant in HEK293T cells. HA-p62 was detected by immunofluorescence with anti-HA (red) antibody. Nuclei were stained with Hoechst stain (scale bar: 5 μm). The experiment was repeated twice. (E) PARP7 promotes STAT1 and STAT2 ubiquitination. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resins, and the ubiquitination of STAT1 or STAT2 was analyzed by western blot. The experiment repeated twice. (F) PARP7 promotes p62 interaction with STAT1 and STAT2. HEK293T cells were co-transfected with FLAG-STAT1 (left) or FLAG-STAT2 (right) and GFP-tagged PARP7 WT or H532A mutant. STAT1 or STAT2 was immunoprecipitated by anti-FLAG affinity resin and blotted for p62. The experiment was repeated twice. (G) p62 knockdown diminishes the difference in STAT1 and STAT2 levels between PARP7 WT and KO MEF cells. STAT1 and STAT2 protein levels in PARP7 WT and KO MEF cells with or without p62 knockdown were analyzed by western blots. The experiment was repeated twice. (H) Proposed model depicting the negative regulation of STAT1 and STAT2 by PARP7. PARP7 binds and ADP-ribosylates STAT1 and STAT2. The ADP-ribosylation recruits E3 ubiquitin ligases, leading to the ubiquitination of STAT1 and STAT2. The ubiquitinated STAT1 and STAT2 then recruits p62, leading to autophagy-mediated degradation. Created with BioRender.com . See also – .

Article Snippet: STAT1 (9172), phospho-STAT1 (Tyr701) (D4A7) (7649), STAT2 (D9J7L) (72604), phospho-Stat2 (Tyr690) (# 4441), ubiquitin (P4D1) (# 3936), TBK1/NAK (3013), phospho-TBK1/NAK (Ser172) (D52C2) (# 5483), SQSTM1/p62 (# 5114), Poly/Mono-ADP Ribose (D9P7Z) (# 89190), JAK1 (6G4) (# 3344), α-Tubulin (# 2144) antibodies were purchased from Cell Signaling.

Techniques: Western Blot, Mutagenesis, Transfection, Immunofluorescence, Staining, Ubiquitin Proteomics, Immunoprecipitation, Knockdown

Journal: Infection and Immunity

Article Title: Legionella pneumophila inhibits type I interferon signaling to avoid cell-intrinsic host cell defense

doi: 10.1128/iai.00365-23

Figure Lengend Snippet: L. pneumophila inhibits cell surface expression of tetherin but does not affect the phosphorylation of STAT1 and STAT2. THP-1 cells were infected with GFP-expressing wild-type L. pneumophila and dotA deficient mutant (Lp∆) at an MOI of 10 and treated or not with 10 ng/mL IFN-β. (A) The flow cytometry analysis performed on the GFP+/infected cells using a fluorescent antibody targeting the cell surface ISG protein tetherin at 20 h.p.i.. (B) The flow cytometry analysis performed on the GFP+/infected cells at an MOI of 10 using a fluorescent antibody targeting phosphorylated STAT1 30 minutes post-infection. (C) The flow cytometry analysis performed on the GFP+/infected cells at an MOI of 10 using a fluorescent antibody targeting phosphorylated STAT2 30 minutes post-infection. The histograms are from one experiment representative of three independent experiments. The bar graphs show the mean fluorescent intensity fold changes compared with the uninfected untreated cells for each flow cytometry analysis. Individual points represent independent experiments. An asterisk indicates multiple unpaired t-tests with Welch correction relative to the uninfected IFN-β treated cells. Significance is indicated as follows: **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Article Snippet: Next, 50 μL of the stain cocktail [1× Brillant Stain Buffer (BD Bioscience) and either 5 μL per sample of Alexa Flour 647 Anti-Total Stat1 (N-Terminus) (BD Biosciences) and 5 μL per sample of PE anti-STAT1 Phospho (Tyr701) (BioLegend) or 2 μL per sample of Stat2 (D9J7L) Rabbit mAb (PE) (Cell Signaling Technology) and 2 μL per sample of P-Stat2 (P-690) Rabbit mAb (Alexa 647) (Cell Signaling Technology)] was added to each sample.

Techniques: Expressing, Phospho-proteomics, Infection, Mutagenesis, Flow Cytometry