Pstat1 y701

MDA-MB-231 human breast cancer cells (American Type Culture Collection, Manassas, VA) were cultured in L-15 Leibovitz's Medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and maintained at 37°C with atmospheric CO₂ according to the suppliers instructions. Purified recombinant cmvIL-10, hIL-10, IFNγ, IL-6 and anti-cmvIL-10, anti-hIL-10, anti-IL-10R, and anti-serpin E1/PAI antibodies were purchased from R&D Systems (Minneapolis, MN). Total Stat3, pStat3 (Y705), total Stat1, and pStat1 (Y701) antibodies were from Cell Signaling Technology (Danvers, MA). The Stat3 inhibitor was from Santa Cruz Biotechnology (Dallas, TX), the Jak1 and p38 MAPK inhibitors were from Calbiochem/EMD Millipore (Billerica, MA). Etoposide was from Cayman Chemicals (Ann Arbor, MI) and purified recombinant human EGF was from Peprotech (Rocky Hill, NJ). The HCMV strain AD169 (ATCC) was propagated in human foreskin fibroblasts (HFF, also from ATCC), maintained in Dulbecco’s modification of Eagle’s medium (Corning) containing 15% fetal bovine serum.

At designated time points after cytokine stimulation, total cell lysates from BMDMs or BMDCs were washed three times with room temperature PBS. For experiments including cytokine stimulation followed by rest, BMDMs or BMDCs were treated for 30 min with designated concentration of cytokine, washed once with room temperature PBS, and cultured in cytokine-free media for specified time of rest. The cells were lysed in the culture dish using 0.02% NP-40 supplemented with HALT protease and phosphatase inhibitors (Thermo Fisher Scientific) and 1× SDS–PAGE buffer (0.0625 M Tris-Cl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-ME, and 0.01% bromophenol blue) was added. Equivalent protein amounts were loaded into 10% acrylamide gels and transferred onto Polyvinylidene difluoride membranes (Millipore). Blots were probed for IFNGR1 (K17), pSTAT1Y⁷⁰¹ (58D6; Cell Signaling), or Total STAT1 (91-C; Cell Signaling) with β-Actin (8H10D10; Cell signaling) as a loading control on each blot. Blots were developed using the secondary antibodies goat α-rabbit IR 800 (926–32211; LI-COR) and goat α-mouse IR 680 (926–68070; LI-COR) and imaged on an Odyssey CLX (LI-COR). All pSTAT1Y⁷⁰¹ and IFNGR1 bands were normalized to β-Actin on the same blot using ImageStudio ver 4.0 software (LI-COR). Densitometry graphs are pooled from at least three independent pSTAT1Y⁷⁰¹ or IFNGR1 blots.

A human ovarian tissue microarray was obtained from Alena Biotechnology Co., Ltd. (Cat# OV1005a, Xi’an, Shanxi, China). All tissues were 10% formalin-fixed and paraffin-embedded. A total of 100 ovarian tissue specimens (20 normal controls and 80 ovarian tumors) were examined by immunohistochemistry (IHC). Among 100 specimens in a slide, seven came off during the IHC staining process. In the end, 20 normal controls (3 from normal ovaries and 17 from adjacent normal ovary tissues) with median age 48.5 years (range 19–63 years) and 73 ovarian tumors (12 benign, 7 borderline, 44 malignant, 10 metastatic) with median age 49.0 years (range 17–75 years) were statistically analyzed.
After blocking with 10% normal goat serum (Fuzhou Maixin Biotech Co., Ltd., Maixin Bio, Fuzhou, Fujian, China), the sections were incubated with rabbit monoclonal antibodies against STAT1 (Cat# 9175), pSTAT1-Y701 (Cat# 9167) and pSTAT1-S727 (Cat# 8826) (Cell Signaling Technology, Inc., Danvers, MA, USA), respectively, overnight, followed by incubation with biotinylated anti-rabbit secondary antibody (Cell Signaling Technology) at room temperature for 1 h. Scoring of STAT1 immunoreactive staining was performed by two independent examiners without any prior view of patient’s clinical data and classified as described previously using staining index (SI) system [21 (link)].

FLSs were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with phosphatase and protease inhibitors (Roche). Protein extracts were separated by electrophoresis, followed by electrotransfer onto nitrocellulose membrane. After blocking, membranes were incubated with primary antibodies and then exposed to horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratory). Specific bands were detected with the enhanced chemiluminescence (ECL) detection kit (Pierce) on Amersham Hyperfilm ECL (GE Healthcare). Reblots were performed using ReBlot Plus Strong Antibody Solution (Millipore). Primary antibodies used were against actin (Cytoskeleton); AKT, p-AKT (S473), p-AKT (T308), p-IKKα/β (S176/180), p-IκB-α (S32/36), IRF1, mTOR, p-mTOR (S2448), p-NF-κB P65 (S536), p-NF-κB P105 (S933), NF-κB P105, p-S6K1 (T389), PTGS2, STAT1, p-STAT1 (Y701), and p-TSC2 (S939) (Cell Signaling Technology); IκB-α, NF-κB P65, and S6K1 (Santa Cruz); SLC38A9 (Sigma); and tubulin (Abcam). FLS lysis and western blotting procedures for SLC38A9 are described elsewhere (Rebsamen et al., 2015 (link)).

The following primary antibodies were used for immunofluorescence and immunoblot analyses as well as coimmunoprecipitation experiments: Stat1 (Cell Signaling, 9172), p-Stat1 S727 (Cell Signaling, 9177), p-Stat1 Y701 (Cell Signaling, 9167), Smad2/3 (Cell Signaling, 5678); Smad3 and p-Smad3 (Cell Signaling [ref. 22 (link)]), SRF (Cell Signaling, 5147), Gata-4 (Cell Signaling, 36966 or Developmental Studies Hybridoma Bank [DSHB]; CRP-Gata-4-1A7, deposited to the DSHB by Protein Capture Reagents Program, produced by JHU/CDI), p-Histone H3 S10 (Cell Signaling, 3377), JAK1 (Cell Signaling, 3332), STAT3 (Cell Signaling, 9404), GAPDH (Santa Cruz Biotechnology, sc-32233), titin T11 (MilliporeSigma, T9030), sarcomeric myosin (DSHB; A4.1025), troponin-I (Cell Signaling, 4002), sarcomeric α-actinin-2 (clone EA-53, Mob 227-05, Diagnostic Biosystems), cardiac actin (Progen Biotechnik, Ac1–20.4.2), PTEN (Cell Signaling, 9188), FoxO1 (Cell Signaling, 2880), normal rabbit IgG (Santa Cruz Biotechnology, sc-2027), normal mouse IgG (Santa Cruz Biotechnology, sc-2025). Secondary fluorescence dye–linked or horseradish peroxidase–linked antibodies were obtained from Jackson Immunoresearch, DAKO, Cell Signaling or Santa Cruz Biotechnology. Fluorescently labeled phalloidin and DAPI was purchased from Molecular Probes (Life Technologies).

Cells were seeded in 6-well plates (50,000 cells/cm²); after 24 h, cells were treated for the indicated times. Cells were then harvested in Laemmli buffer, boiled for 5 min at 95 °C and purified with the NucleoSpin Filters (Macherey–Nagel GmbH, Düren, Germany). Samples were run on SDS–PAGE. Transfer on nitrocellulose membranes was performed by using iBlot™ 2 Dry Blotting System (Invitrogen). Membranes were then incubated in 5% milk in T-TBS for 1 h and overnight with the primary antibody in 5% BSA in T-TBS at 4 °C (p-STAT1 Y701, Cell signaling technology). After washing steps, membranes were incubated with secondary antibody conjugated with horseradish peroxidase (GE Healthcare Bio-Sciences©) for 1 h at room temperature. Signal was finally detected using Pierce ECL wester blotting substrate (Thermo Scientific© Boston, MA, USA) and imaged with a Fusion-SL 3500 WL device and Fusion software (Bio-Rad™, Hercules, CA, USA). Afterwards, the membrane was further incubated for 3 h with the primary antibody specific for the housekeeping protein GAPDH (Invitrogen). The same procedure as described above was followed for detection and imaging of the housekeeping signal.