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2 protocols using socs1

1

Quantifying Immune Response Transcripts in Neutrophils

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Total RNA was isolated using the RNeasy Mini kit following the manufacturer’s instructions (Qiagen, Hilden, Germany). RNA quality and quantification were determined by TapeStation 4200 (Agilent, St. Clara, USA) following the manufacturer’s instructions. Neutrophil total RNA was isolated using RNeasy Mini kit following the manufacturer’s instructions (Qiagen), and complementary DNA (cDNA) was synthesized using M-MLV Reverse Transcriptase (Thermo Fisher Scientific). RT-PCR was performed in duplicates using the cDNA and Platinum Taq polymerase (Thermo Fisher Scientific), 200 nM dNTP (Promega, Southampton, UK), 50mM MgCl2 (Thermo Fisher Scientific), and TaqMan primer/probe sets (Thermo Fisher Scientific). Samples were matched to a standard curve generated by amplifying serially diluted products using the same PCR reaction and normalized to GAPDH (Hs00266705_g1) to obtain the relative expression value. Real-time assays were run on FX96 Cycler (Bio-Rad). The following are the primes used: STAT1 (Hs01013996_m1), STAT2 (Hs01013115_g1), STAT3 (Hs00374280_m1), IFIH1 (Hs00223420_m1), IFIT (Hs00356631_g1), ISG15 (Hs00192713_m1), MX1 (Hs00895608_m1), IRF1 (Hs00971965_m1), SOCS1 (Hs00705164_s1), USP18 (Hs00276441_m1), IFI44 (Hs00197427_m1), IFI16 (Hs00986757_m1), and OAS (Hs00242943_m1) (all from Thermo Fisher Scientific).
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2

Immunoblotting of JAK-STAT Signaling Proteins

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Whole-cell extracts for immunoblotting were prepared by incubating cells for 20 min in RIPA lysis buffer (Thermo Fisher Scientific) with 50 mM dithiothreitol and Protease/Phosphatase inhibitor cocktail (Cell Signaling Technology). Protein was quantified with Micro BCA Protein Assay (Thermo Fisher Scientific). Immunoblotting was performed using the Bio-Rad Western blot workflow. Membranes were blocked in 5% BSA for primary antibodies or 5% nonfat dry milk for secondary antibodies. Antibodies used were STAT1 (Santa Cruz Biotechnology), STAT2 (Millipore), phospho-Tyr 701 STAT1 (Cell Signaling Technology), phospho-Tyr 689 STAT2 (Cell Signaling Technology), USP18 (Cell Signaling Technology), IFIT1 (Cell Signaling Technology), MX1 (Abcam), SOCS1 (Thermo Fisher Scientific), β-actin (ABclonal), and GAPDH (Millipore). Signal was detected with enhanced chemiluminescence detection reagent (ECL or SuperSignal West pico, Thermo Fisher Scientific) by film development or capturing on ImageQuant 800 Fluor imaging system (Cytiva). For all representative immunoblots shown, n=3 separate experiments were carried out.
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