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The IFI44 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is designed to perform a specific function, but without more information, a detailed and unbiased description cannot be provided while maintaining conciseness. A comprehensive and factual description is not available at this time.

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4 protocols using ifi44

1

qPCR Analysis of Immune Genes

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qPCR was performed using the TaqMan Gene Expression Master Mix (Applied Biosystems, Thermo Fisher Scientific, 4369016). Data were analyzed by the 2-DDCt method using the mean adult values as the reference. Primers/probes were from Thermo Fisher Scientific: MX1 (Hs00895608_m1), MX2 (Hs01550811_m1), IFNA1 (Hs00256882_s1), IFI44 (Hs00951349_m1), IFIT1 (Hs03027069_s1), IL17A (Hs00174383_m1), IFNG (Hs00989291_m1), RPLPO (4326314E).
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2

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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3

Quantifying Immune Gene Expression

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Total RNA was extracted from cells (1–2 × 106 cells per sample) with an RNeasy kit (Qiagen, Germantown, MD, USA) and cDNA was generated using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). qRT-PCR was performed on a Light Cycler 480 II (Roche, Indianapolis, IN, USA). Primers for IFIT1 (Hs03027069), IFI44 (Hs00197427), RAGE (Hs00542584), FcRIIa (Hs01013401), MX1 (Hs00182073,), HPRT1 (Hs99999909), and Polr2a (Hs00172187) were purchased from Thermo Scientific. The genes of interest were normalized to the expression of the house keeping gene (Polr2a) and were compared to a control condition with no treatment. The relative induction was calculated by 2−ΔΔCt.
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4

Western Blot Analysis of TNBC PDX Tumors

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About 50 mg of frozen TNBC PDX tumors were used to extract total protein with T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s guidelines. The protein concentration was evaluated by Bradford Assay. The equal amount of denatured protein extracts was separated through electrophoresis in 4–15% NuPAGE Tris-HCl precast gels (Invitrogen, Waltham, MA, USA) and transferred onto nitrocellulose membranes. Membranes were then probed with primary antibodies including Cd74, Sat1, TAp63, Bcl-xL, Dnmt1, Dnmt3a, Dnmt3b, Tet2, Tet3, Hdac1, Hdac2, Hdac3, Hdac8 (Cell Signaling Technology, Danvers, MA, USA), Tet1, Ifi44, Fzd9, Wwc1 (Thermo Fisher Scientific, Waltham, MA, USA) and NF-κB and Lpl (Santa Cruz, Dallas, TX, USA). β-actin served as an internal control for each membrane. Immunoreactive bands were visualized using Clarity MaxTM Western ECL Blotting Substrates (Bio-Rad, Hercules, CA, USA) and images were captured using ChemiDocTM Imaging Systems (Bio-Rad, Hercules, CA, USA). The protein expression levels were quantified using Image J software (v1.53e).
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