Pstat1 tyr701

After induction with IL-10 (30 ng/ml) for the indicated time points, 2 × 10⁵ cells were lysed in 80 µl of 2x SDS sample buffer (Bio-Rad), separated on a precast NuPAGE 4–12% gradient gel (Life Technologies) and transferred onto a nitrocellulose membrane (Bio-Rad). Blocking was performed for 1 h in TBS containing 0.1% Tween 20 and 5% nonfat dry milk. Antibodies against p-STAT1-Tyr701 (Cat no. 9167), STAT1 (Cat no. 9172), p-STAT3-Tyr705 (Cat no. 9145), STAT3 (Cat no. 4904), β-Actin (Cat no. 4970), SOCS1 (Cat nos 3957 and 3950), SOCS2 (Cat no. 2779), SSH1 (Cat no. 13578) and HRP-linked anti-rabbit secondary antibody (Cat no. 7074) were all purchased from Cell Signaling Technology and used according to the manufacturer’s instructions. West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) and BioMax film (Kodak) were used for detection. ImageJ (NIH) was used for quantification of Western Blots.

Cells were lysed directly with SDS sample buffer (4% sodium dodecyl sulfate [SDS], 20% glycerol, 50 mM Tris HCl (pH 6.8)), proteins separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane. Membranes were incubated overnight with specific primary antibodies diluted in blocking buffer. These included: TRAIL, PUMA, AKT, phospho-AKT(Ser473), phospho-p70S6K1(Thr389), ERKp44/p42, phospho-ERK(Thr202/Tyr204) and p-STAT1(Tyr701) from Cell Signaling Technology; MCL-1 and GAPDH from Santa Cruz Biotechnology; BCL-2 from DaKo; ISG15, ISG54, OAS1 and PML from Proteintech; β-actin from Millipore. After washing, bound antibodies were detected with fluorescent-conjugated secondary anti-rabbit or anti-mouse antibodies (Enzo Life Sciences), then visualized and quantified with an Odyssey system (Pierce, Waltham, MA, USA).

The PDX tissues were ground in liquid nitrogen and then were lysed. Next, protein was extracted by centrifugation for 30 min at 12,000 rpm. Protein (50 μg) was separated in 10% SDS-PAGE gel, and transferred to PVDF membrane at 90 V for 2 h. The membrane was blocked with 5% no-fat milk for 60 min. Later, membrane was incubated with mTOR, p-mTOR (Ser2481), STAT1, p-STAT1 (Tyr701), STAT3, p-STAT3 (Tyr705), AKT1, p-AKT (Ser473), ERK, p-ERK (Thr202/Tyr204) antibodies (Cell Signaling Technology, America) overnight at 4 °C. All antibodies were used at 1:1000. The STAT3 antibody was from mouse, other antibodies were from rabbit. The fluorescent secondary antibody was incubated at room temperature for 1.5 h. The PVDF membranes were scanned by Odyssey and analyzed by Image Studio Ver 2.1.

Cells were washed with PBS and lysed in 1x RIPA buffer with protease and phosphatase inhibitors, with the addition of 1 U/ml Benzonase to degrade genomic DNA. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 hr at RT in LiCOR Odyssey blocking buffer. Blots were (Licor) and incubated overnight at RT with the following antibodies: IRF3 (Cell Signaling, 1:1000); pIRF3 Ser396 (Cell Signaling, 1:1000); STAT1 (Cell Signaling, 1:1000); pSTAT1 Tyr701 (Cell Signaling, 1:1000); Beta Actin (Abcam, 1:2000), β-Tubulin (Abcam, 1:5000); DRP1 (Cell Signaling, 1:1000); pDRP1 Ser616 (Cell Signaling, 1:1000), TFAM (Millipore, 1:1000), VDAC (Protein Tech, 1:1000). Membranes were incubated with appropriate secondary antibodies for 2 hr at RT prior to imaging on a LiCOR Odyssey Fc Dual-Mode Imaging System.

Mouse proteins were separated by SDS-PAGE and transferred to PVDF membrane. After blocking in TBS-T with 1% BSA in (TBS-T), the blots were incubated with primary antibody (1:1000 dilution) overnight. The rabbit monoclonal antibodies for LC3 (#12741), STAT1 (#9172), pSTAT1-Tyr701 (#9167), NFκB phospho-p65 (Ser536) (#3033), p62/SQSTM1 (#23214) and NFκB p65 (#8242) were obtained from Cell Signaling. The anti-NKLAM antibody has been described previously [6 (link)]. The blots were washed 3 times in TBS-T and incubated with horse radish peroxidase-conjugated secondary antibodies. The images were captured with a BioRad Chemidoc XRS+ imager. Protein normalization was performed using β-actin Western blot or with 2,2,2-trichloroethanol (TCE). Briefly, TCE (54 mM final concentration) was added to the polyacrylamide gel solution prior to polymerization. TCE binds to tryptophan residues and becomes fluorescent when exposed to UV light [14 (link)], allowing the visualization of the total protein in each lane. For normalization, the band intensity for a protein of interest was divided by the intensity of the TCE signal in the corresponding lane. For phosphoprotein analysis (pSTAT1 and pp65), the band intensity of the phosphorylated form of the protein was divided by the band intensity of the total protein.

For western blotting, cells were resuspended in lysis buffer containing protease inhibitor cocktail (Roche, Germany) and PhosSTOP (Roche). Gel elecrophoresis was performed using 10% polyacrylamide gels, and blotting was performed using the Trans-blot Turbo Transfer System (Bio-rad, The Netherlands) and the Trans-blot turbo RTA Transfer kit (LF PVDF, Bio-rad). Antibodies used were the following: ERK1/2 (137F5; 4695; Cell Signaling, The Netherlands), P-ERK1/2 (E-4;SC-7383; Santa Cruz Biotechnology, Texas), JNK1/2 (9252; Cell Signaling), P-JNK1/2 (81E11; 4668; Cell Signaling), P38 (9212; Cell Signaling), P-P38 (9211; Cell Signaling), P-STAT1 (Tyr-701; 9167; Cell Signaling), α-Tubulin (Cedarlane, Canada) and PARP1 (4C10-5, BD Biosciences, The Netherlands). As secondary antibodies we used Goat-anti Rabbit IRDye 800CW/680RD (LI-COR Biosciences, Nebraska), Goat-anti-mouse IRDye 800CW/680RD (LI-COR Biosciences). Imaging was performed on the Odyssey (LI-COR Biosciences), and quantification of signal intensity was done using Image Studio (LI-COR Biosciences). The signal intensity of phosphoproteins was corrected for the signal intensity of the (total) unphosphorylated protein (ERK/P38/JNK) or for the signal intensity of a housekeeping gene (α-Tubulin to correct for STAT1α/β).

Cells were collected and total protein was extracted for protein quantification by BCA. After 20 μg protein was subjected to SDS-PAGE and transferred to polyvinylidene fluoride membranes (millipore, USA), the corresponding primary antibodies against phosphorylated signal transducer and activator of transcription (p-STAT)1-tyr⁷⁰¹, p-STAT6-tyr⁶⁴¹ (Cell Signaling Technology, Danvers, MA, USA) and β-actin (Sigma, USA) were applied at 4 °C overnight. Then the membranes were washed and incubated with HRP-conjugated secondary antibodies with shaking at room temperature for 30 min. Immunoreactive proteins were exposed and developed using enhanced chemiluminescence (Beyotime, China), and β-actin was used as an internal reference to calculate the relative expression of proteins.