anti pstat1 cell signaling technology 9167 wb rabbit hu 58d6  (Cell Signaling Technology Inc)

Journal: Cell

Article Title: Methyltransferase SETD2-Mediated Methylation of STAT1 Is Critical for Interferon Antiviral Activity.

doi: 10.1016/j.cell.2017.06.042

Figure Lengend Snippet: Figure 3. SETD2 Facilitates IFNa-Induced STAT1 Phosphorylation and ISGs Expression (A) IB analysis of phosphorylated (p)STAT1, pSTAT2, SETD2, STAT1, STAT2, H3K36me3, and Histone3 in lysates of HepG2 cells transfected with si-NC or si-SETD2 followed by 1,000 U/mL IFNa treatment for the indicated time. GAPDH was used as a control. (B) IB analysis of pSTAT1, pSTAT2, STAT1, and STAT2 in cytoplasmic and nuclear extracts of HepG2 cells transfected and stimulated as in (A). GAPDH and Lamin A/C were used as the cytoplasmic and nuclear controls, respectively.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-pSTAT1 Cell Signaling Technology Cat #9171; RRID: AB_331591 anti-STAT1 Cell Signaling Technology Cat #9172; RRID: AB_10831362 anti-flag Cell Signaling Technology Cat #2368; RRID: AB_2217020 anti-IRF9 Cell Signaling Technology Cat #76684 anti- GST Cell Signaling Technology Cat #2625; RRID: AB_490796 anti-histone 3 Abcam Cat #ab1971 anti-H3K36me3 Abcam Cat #ab9050; RRID: AB_306966 anti-lamin A/C Abcam Cat #ab108595; RRID: AB_10866185 anti- HBcAg Abcam Cat #ab115992; RRID: AB_10903422 anti-GAPDH Proteintech Cat #HRP-60004 anti-STAT2 Millipore Cat #07-140; RRID: AB_11213767 anti-pSTAT2 Millipore Cat #07-224; RRID: AB_2198439 anti-K525me1 ABmart Cat #26703-1hV anti-SETD2 Abclonal Cat #A3194 FITC-conjugated anti-mouse IgG antibody BD Bioscience Cat #555988; RRID: AB_396275 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins Human Interferon Alpha 2 (Alpha 2b) Pestka Biomedical Laboratories Cat #11105-1 Mouse Interferon Alpha A Pestka Biomedical Laboratories Cat #12100-1 Recombinant Human IFN-b PeproTech Cat #300-02BC Recombinant Human IFN-g PeproTech Cat #300-02 Recombinant Human IFN-l1 PeproTech Cat #300-02L Recombinant Human M-CSF PeproTech Cat # 300-25 APC-conjugated Annexin V BD Bioscience Cat # 550474 propidium iodide BD Bioscience Cat # 556463 Critical Commercial Assays human HBsAg ELISA kit Kehua Biotechnology (http://www.skhb.com/cn/) Cat # S10910113 GST Protein Interaction Pull-Down Kit Thermo Cat # 21516 LightShift EMSA Optimization and Control Kit Thermo Cat # 20148X ChIP assay kit Millipore Cat # 17-371FR Deposited Data RNA-seq raw data This paper GEO: GSE98372 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A HepG2.2.15 Cell Bank of the Chinese Academy of Sciences N/A BHK-21 ATCC Cat # CCL-10 HEK293t ATCC Cat # CRL-11268 (Continued on next page) Cell 170, 492–506.e1–e6, July 27, 2017 e1

Techniques: Phospho-proteomics, Expressing, Transfection, Control

Journal: Cell

Article Title: Methyltransferase SETD2-Mediated Methylation of STAT1 Is Critical for Interferon Antiviral Activity.

doi: 10.1016/j.cell.2017.06.042

Figure Lengend Snippet: Figure 5. Methylated Lysine 525 of STAT1 Is Necessary for Its Phosphorylation and Transcriptional Activity (A) Identification of methylated lysine residues in STAT1 in HepG2 cells by immunoprecipitation combined with mass spectrometry. (B) Tandem mass spectrometry of STAT1 peptide modified with methylation on lysine 525 residue. (C) IB analysis of pSTAT1 and total STAT1 in whole-cell lysates of STAT1-KO HepG2 cells transfected with wild-type STAT1 (STAT1-WT) or point mutated STAT1 expression plasmids, followed by 1,000 U/mL IFNa treatment for 6 hr. (D) IB analysis of pSTAT1 and total STAT1 in nuclear and cytoplasmic (cyto) extracts of STAT1-KO cells transfected with STAT1-WT or STAT1-K525A expression plasmids and stimulated as in (C). (E) qRT-PCR analysis of ISG15, IFIT-1, and MX2 expression in STAT1-KO cells treated as in (C). (F) IB analysis of immunoprecipitates of STAT1-KO HepG2 cells co-transfected with myc-tagged JAK1 and flag-tagged STAT1-WT or STAT1-K525A expression plasmids followed by IFNa treatment for 30 min. (G) EMSA of whole-cell lysates from STAT1-KO cells transfected as in (C) and treated with IFNa for indicated time. 13 ISRE motif and 13 GAS motif were biotin- labeled. Data are representative photographs of three independent experiments with similar results or shown as mean ± SD of three independent experiments (E). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S4 and S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-pSTAT1 Cell Signaling Technology Cat #9171; RRID: AB_331591 anti-STAT1 Cell Signaling Technology Cat #9172; RRID: AB_10831362 anti-flag Cell Signaling Technology Cat #2368; RRID: AB_2217020 anti-IRF9 Cell Signaling Technology Cat #76684 anti- GST Cell Signaling Technology Cat #2625; RRID: AB_490796 anti-histone 3 Abcam Cat #ab1971 anti-H3K36me3 Abcam Cat #ab9050; RRID: AB_306966 anti-lamin A/C Abcam Cat #ab108595; RRID: AB_10866185 anti- HBcAg Abcam Cat #ab115992; RRID: AB_10903422 anti-GAPDH Proteintech Cat #HRP-60004 anti-STAT2 Millipore Cat #07-140; RRID: AB_11213767 anti-pSTAT2 Millipore Cat #07-224; RRID: AB_2198439 anti-K525me1 ABmart Cat #26703-1hV anti-SETD2 Abclonal Cat #A3194 FITC-conjugated anti-mouse IgG antibody BD Bioscience Cat #555988; RRID: AB_396275 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins Human Interferon Alpha 2 (Alpha 2b) Pestka Biomedical Laboratories Cat #11105-1 Mouse Interferon Alpha A Pestka Biomedical Laboratories Cat #12100-1 Recombinant Human IFN-b PeproTech Cat #300-02BC Recombinant Human IFN-g PeproTech Cat #300-02 Recombinant Human IFN-l1 PeproTech Cat #300-02L Recombinant Human M-CSF PeproTech Cat # 300-25 APC-conjugated Annexin V BD Bioscience Cat # 550474 propidium iodide BD Bioscience Cat # 556463 Critical Commercial Assays human HBsAg ELISA kit Kehua Biotechnology (http://www.skhb.com/cn/) Cat # S10910113 GST Protein Interaction Pull-Down Kit Thermo Cat # 21516 LightShift EMSA Optimization and Control Kit Thermo Cat # 20148X ChIP assay kit Millipore Cat # 17-371FR Deposited Data RNA-seq raw data This paper GEO: GSE98372 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A HepG2.2.15 Cell Bank of the Chinese Academy of Sciences N/A BHK-21 ATCC Cat # CCL-10 HEK293t ATCC Cat # CRL-11268 (Continued on next page) Cell 170, 492–506.e1–e6, July 27, 2017 e1

Techniques: Methylation, Phospho-proteomics, Activity Assay, Immunoprecipitation, Mass Spectrometry, Residue, Transfection, Expressing, Quantitative RT-PCR, Labeling

Journal: Cell

Article Title: Methyltransferase SETD2-Mediated Methylation of STAT1 Is Critical for Interferon Antiviral Activity.

doi: 10.1016/j.cell.2017.06.042

Figure Lengend Snippet: Figure 6. SETD2 Directly Mediates STAT1 K525me1 (A) IB analysis of mono-methylation of STAT1, pSTAT1, total STAT1, and GAPDH in immunopre- cipitates, and whole-cell lysates of STAT1-KO cells transfected with flag-tagged STAT1-WT or STAT1-K525A expression plasmids followed by 1,000 U/mL IFNa treatment for 6 hr. (B) IB analysis of mono-methylation of STAT1 and total STAT1 in immunoprecipitates of STAT1-KO cells co-transfected with flag-tagged SETD2-F2 and myc-tagged STAT1-WT or STAT1-K525A expression plasmids stimulated with IFNa as in (A). (C) IB analysis of mono-methylation on K525 (K525me1) of STAT1, total STAT1 in immuno- precipitates of STAT1-WT, and STAT1-K525A- rescued STAT1-KO cells stimulated with IFNa as in (A). (D) IB analysis of STAT1 K525me1, pSTAT1, total STAT1, and GAPDH in immunoprecipitates and the whole-cell lysates of HepG2 cells transfected with si-NC or si-SETD2, stimulated with IFNa as in (A). (E) IB analysis of STAT1-525me1 in the in vitro methylation assay with purified 63 his-tagged STAT1 and GST-tagged SETD2 truncation (1469– 1724 aa) using S-adenosyl-L-methionine (SAM) as the methyl group donor. (F and G) IB analysis of JAK1, STAT2, IRF9, and STAT1 in immunoprecipitates and the whole-cell lysates of WT and SETD2-KO HepG2 cells treated with IFNa (F) or IFNb (G) for 30 min. Data are shown for one representative experiment. Similar results were obtained in three independent experiments. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies anti-pSTAT1 Cell Signaling Technology Cat #9171; RRID: AB_331591 anti-STAT1 Cell Signaling Technology Cat #9172; RRID: AB_10831362 anti-flag Cell Signaling Technology Cat #2368; RRID: AB_2217020 anti-IRF9 Cell Signaling Technology Cat #76684 anti- GST Cell Signaling Technology Cat #2625; RRID: AB_490796 anti-histone 3 Abcam Cat #ab1971 anti-H3K36me3 Abcam Cat #ab9050; RRID: AB_306966 anti-lamin A/C Abcam Cat #ab108595; RRID: AB_10866185 anti- HBcAg Abcam Cat #ab115992; RRID: AB_10903422 anti-GAPDH Proteintech Cat #HRP-60004 anti-STAT2 Millipore Cat #07-140; RRID: AB_11213767 anti-pSTAT2 Millipore Cat #07-224; RRID: AB_2198439 anti-K525me1 ABmart Cat #26703-1hV anti-SETD2 Abclonal Cat #A3194 FITC-conjugated anti-mouse IgG antibody BD Bioscience Cat #555988; RRID: AB_396275 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins Human Interferon Alpha 2 (Alpha 2b) Pestka Biomedical Laboratories Cat #11105-1 Mouse Interferon Alpha A Pestka Biomedical Laboratories Cat #12100-1 Recombinant Human IFN-b PeproTech Cat #300-02BC Recombinant Human IFN-g PeproTech Cat #300-02 Recombinant Human IFN-l1 PeproTech Cat #300-02L Recombinant Human M-CSF PeproTech Cat # 300-25 APC-conjugated Annexin V BD Bioscience Cat # 550474 propidium iodide BD Bioscience Cat # 556463 Critical Commercial Assays human HBsAg ELISA kit Kehua Biotechnology (http://www.skhb.com/cn/) Cat # S10910113 GST Protein Interaction Pull-Down Kit Thermo Cat # 21516 LightShift EMSA Optimization and Control Kit Thermo Cat # 20148X ChIP assay kit Millipore Cat # 17-371FR Deposited Data RNA-seq raw data This paper GEO: GSE98372 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A HepG2.2.15 Cell Bank of the Chinese Academy of Sciences N/A BHK-21 ATCC Cat # CCL-10 HEK293t ATCC Cat # CRL-11268 (Continued on next page) Cell 170, 492–506.e1–e6, July 27, 2017 e1

Techniques: Methylation, Transfection, Expressing, In Vitro