Journal: Molecular Metabolism

Article Title: Baricitinib counteracts metaflammation, thus protecting against diet-induced metabolic abnormalities in mice

doi: 10.1016/j.molmet.2020.101009

Figure Lengend Snippet: Baricitinib attenuates the HD-induced JAK-STAT pathway. Western blotting analysis for phosphorylation of Tyr 1007/1008 JAK1/2 in the skeletal muscle (A) and kidneys (B) and normalized to total JAK1/2 and for phosphorylation of Tyr 690 on STAT2 in the skeletal muscle (C) and kidneys (D) and normalized to total STAT2. All of the data are expressed as mean ± SEM for n = 6 per group. ∗p< 0.05 vs ND and •p< 0.05 vs HD.

Article Snippet: The antibodies used were rabbit anti-Tyr 1007/1008 JAK2 (#3776), rabbit anti-total JAK2 (#3230), rabbit anti-Tyr 690 STAT2 (Bioss Antibodies, bs-3428R), rabbit anti-total STAT2 (#72604), rabbit p21 (#2947), rabbit anti-Ser 307 IRS-1 (#2381), mouse anti-total IRS-1 (#3194), rabbit anti-Ser 473 AKT (#4060), rabbit anti-total AKT (#9272), rabbit anti-Ser 9 GSK-3β (#9332) and rabbit anti-total GSK-3β (9315).

Techniques: Western Blot

Journal: Science advances

Article Title: N-MYC impairs innate immune signaling in high-grade serous ovarian carcinoma.

doi: 10.1126/sciadv.adj5428

Figure Lengend Snippet: Fig. 3. N-MYC represses IFN type I immune signaling. (A) qRT-PCR of IFN type I (IFNA2 and IFNB1) and III (IFNL1, IFNL2, and IFNL3) ligands in CaOV3 MYCN/GFP and GFP cells treated ± DOX (1 μg/ml) for 72 hours. P values were calculated using an unpaired two-tailed Student’s t test. (B) qRT-PCR of IFNAR1 and IFNLR1 (top) and ISGs (bottom) in CaOV3 wild-type cells transfected with scrambled negative control siRNA or siRNAs specific for IFNAR1 and IFNLR1 for 72 hours. (C) Immunoblot of P-STAT1, STAT1, P-STAT2, STAT2, IRF9, and α-tubulin levels in CaOV3 MYCN/GFP and CaOV3 GFP cells treated ± DOX for 24 and 72 hours. Data are representative of three independent experiments. Paired comparisons are shown in the same color for densitometry fold changes. (D) Immunoblot with the indicated antibodies in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX for 72 hours and then treated with IFNB1 (10 ng/ml) for 2 hours. Nuclear and cytoplasmic fractions were prepared and subjected to Western blot. Data are representative of three independent experiments. (E) ChIP-qPCR analysis of IRF9 binding to the ISRE sequence of the ISG15 promoter in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX for 72 hours and then treated with IFNB1 (50 ng/ml) for 30 min. P values were calculated using one-way analysis of variance (ANOVA) with Tukey posttest for pairwise comparison. (F) qRT-PCR of multiple ISGs in CaOV3 MYCN/GFP and GFP cells pretreated ± DOX (1 μg/ml) for 72 hours followed by IFNB1 pulse (10 ng/ml) for 2 hours and then 24-hour chase. P values were calculated using one-way ANOVA with Tukey posttest for pairwise comparison. Data in (A), (B), (E), and (F) are means ± SEM of n = 3 biological replicates. *P < 0.05; **P < 0.005; ***P < 0.001; ****P < 0.0001. Densitometry analysis was performed using ImageJ software.

Article Snippet: The following antibodies were used in this study: anti- Hu Fc receptor binding inhibitor (#50- 112- 9053, eBioscience), P- STAT1 (Tyr701) (#9167, Cell Signaling Technology), STAT1 (#14994, Cell Signaling Technology), P- STAT2 (Tyr690) (#4441, Cell Signaling Technology), STAT2 (#72604, Cell Signaling Technology), IRF9 (#76684, Cell Signaling Technology), α- tubulin (#3873, Cell Signaling Technology), DYKDDDDK Tag rabbit monoclonal antibody (mAb) (#14793, Cell Signaling Technology), DYKDDDDK Tag mouse mAb (#8146, Cell Signaling Technology), STING (#13647, Cell Signaling Technology), TATA box–binding protein (#8515, Cell Signaling Technology), CD3- PECy7 (#341111, BD Bioscience Technology), RIG- I (#3743, Cell Signaling Technology), MDA- 5 (#5321, Cell Signaling Technology), P- TBK1 (Ser172) (#5483, Cell Signaling Technology), TBK1 (#3504, Cell Signaling Technology), P- IRF3 (Ser396) (#4947, Cell Signaling Technology), N- MYC (#51705, Cell Signaling Technology), VDAC (#4661, Cell Signaling Technology), MAVS (#24930, Cell Signaling Technology), c- MYC (#5605, Cell Signaling Technology), L- MYC (#76266, Cell Signaling Technology), P- STING (Ser366) (#50907, Cell Signaling Technology), hemagglutinin tag (#3724, Cell Signaling Technology), Myc tag (#2278, Cell Signaling Technology), cGAS (#15102, Cell Signaling Technology), anti- rabbit immunoglobulin G (IgG) (H+L) (DyLight 800 4× polyethylene glycol conjugate) (#5151, Cell Signaling Technology), and anti- mouse IgG (H+L) (DyLight 680 conjugate) (#5470, Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Two Tailed Test, Transfection, Negative Control, Western Blot, ChIP-qPCR, Binding Assay, Sequencing, Comparison, Software

Journal:

Article Title: Sendai Virus Blocks Alpha Interferon Signaling to Signal Transducers and Activators of Transcription

doi:

Figure Lengend Snippet: Poor induction of ISG products in SeV-infected cells. HeLa cells were mock infected (lanes 1 to 3) or infected with SeV (lanes 4 to 9), and then the media were replaced at 2 hpi with fresh medium containing 103 IU of IFN-α per ml (lanes 2, 3, 5, and 6) or no IFN-α (lanes 1, 4, 7, 8, and 9). The cells were harvested at 0 (lanes 1, 4, and 7), 8 (lanes 2, 5, and 8), and 24 (lanes 3, 6, and 9) h after IFN-α treatment. Total-cell extracts (20 μg of protein) prepared according to the method of Lee et al. (14) were subjected to Western blot analysis as described previously (9). Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody. The same blotting membrane was stripped and reprobed. The protein concentration was determined as described previously (9).

Article Snippet: Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody.

Techniques: Infection, Western Blot, Protein Concentration

Journal:

Article Title: Sendai Virus Blocks Alpha Interferon Signaling to Signal Transducers and Activators of Transcription

doi:

Figure Lengend Snippet: Inhibition of IFN-α-stimulated tyrosine phosphorylation of Stat1, Stat2, and Stat3 in SeV-infected cells. HeLa cells were mock infected (lanes 1 to 3) or infected (lanes 4 to 9) with SeV at 2 h prior to replacement with fresh medium containing 103 IU of IFN-α per ml (lanes 2, 3, 5, and 6) or no IFN-α (lanes 1, 4, 7, 8, and 9). The cells were harvested at 0 (lanes 1, 4, and 7), 5 (lanes 2, 5, and 8), or 30 (lanes 3, 6, and 9) min after IFN-α treatment. Total-cell extracts (50 μg of protein) were subjected to Western blot analysis with anti-phospho-(Tyr 701)-Stat1 (no. 9171) (A) and anti-phospho-(Tyr 705)-Stat3 (no. 9131) (C) rabbit polyclonal antibodies (New England Biolabs, Inc.). To detect tyrosine-phosphorylated Stat2, total-cell extracts (500 μg) were immunoprecipitated with an anti-Stat2 antibody (sc-476) (B) before Western blot analysis with antiphosphotyrosine mouse monoclonal antibody (sc-7020) (Santa Cruz Biotechnology, Inc.). Each blotting membrane was stripped and reprobed with anti-Stat1 (sc-464) (A) mouse monoclonal antibody or anti-Stat2 (sc-476) (B) or anti-Stat3 (sc-7179) (C) rabbit polyclonal antibody (Santa Cruz Biotechnology, Inc.).

Article Snippet: Anti-PKR rabbit polyclonal (sc-707) (A), anti-Stat1 mouse monoclonal (sc-464) (B), anti-Stat2 rabbit polyclonal (sc-476) (C), and anti-p48 rabbit polyclonal (sc-496) (D) antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used as the first antibody.

Techniques: Inhibition, Infection, Western Blot, Immunoprecipitation

Journal: eLife

Article Title: Multiple tumor suppressors regulate a HIF-dependent negative feedback loop via ISGF3 in human clear cell renal cancer

doi: 10.7554/eLife.37925

Figure Lengend Snippet:

Article Snippet: Antibody , STAT2 (rabbit polyclonal) , Bethyl A303-512A , , , , (1:1,000 for western blot, 1:25 for IHC).

Techniques: Western Blot, Recombinant