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6 protocols using sc 1616

1

Protein Interaction and Cytokine Quantification

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Western blotting and coimmunoprecipitation were performed as described previously using specific antibodies against aromatase (#677; kindly provided by Dr. Dean P. Edwards (Baylor College of Medicine), and sc-14245, Santa Cruz Biotechnology), IFI16 (sc-8023, Santa Cruz Biotechnology), Actin (sc-1616), HIF1α (sc-10790), PRMT2 (ab154154, Abcam), and Ifi204 (NBP2-27153, Novus Biologicals) [49 (link)]. To detect protein–protein interactions with high selectivity and sensitivity, PLAs were performed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma Aldrich) according to manufacturer’s protocol. Briefly, cells were fixed and incubated with primary antibodies against IFI16 (sc-8023, Santa Cruz Biotechnology), HIF1α (sc-10790), and PRMT2 (ab154154, Abcam). The cells were incubated with PLA probes and the subsequent ligation and rolling circle amplification were performed. The PLA signals were visualized using a Zeiss LSM 710 confocal microscope (Carl Zeiss).
The amounts of IFNα, IFNβ, and IFNγ protein were measured using commercial ELISA kits (MBS2506739, MBS2513798, and MBS2512904, respectively, MyBioSource) according to the manufacturer’s protocol. The amount of estradiol was measured using Estradiol ELISA kit (#582251 and #501890, Cayman) according to the manufacturer’s protocol.
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2

Quantitative Western Blot Analysis

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Both procedures have been described in detail in our previous publication [25 (link)]. The following primary antibodies were used for western blotting: anti-HAdV capsid (1:5000; Abcam, Cambridge, UK, ab6982), anti-actin (1:1000; Santa Cruz, sc-1616), anti-eIF2 (1:500, Abcam, ab26197), and anti-eIF2α(P-Ser51) (1:500, Abcam, ab32157). The 35S-signals were detected with Pharos FXTM Plus Molecular Imager and analysed using Quantity One 4.6.9 (Bio-Rad).
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3

Protein Extraction and Western Blot

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Tissues and cultured cells were homogenized in a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 0.05% Triton X-100, 1 mM dithiothreitol (DTT), 1 mM EDTA, and protease inhibitor and phosphatase inhibitor cocktails (Complete mini and phoSTOP phosphatase inhibitor cocktail tablets; Roche Applied Science). Homogenates were cleared by centrifugation at 10,000g for 10 min at 4°C, supernatants were collected, and protein concentration was estimated using Bio-Rad DC protein assay (Bio-Rad) according to the manufacturer's instructions. Total protein was resolved on an 8%–10% SDS–polyacrylamide gel by electrophoresis. Thereafter, proteins were electrotransferred onto a nictrocellulose transfer membrane. The membranes were blocked with PBST (PBS with 0.1% Tween-20) containing 5% powdered milk for 1 h. Anti-PER2 and CRY2 antibodies were a generous gift from Dr. C. Lee, anti-β-ACTIN was from Santa Cruz Biotechnology (sc-1616), anti-NFκB p65 was from Abcam (ab7970), and anti-guinea pig IgG secondary antisera horseradish peroxidase was from Jackson ImmunoResearch. Proteins were visualized with a chemiluminescence detection system with subsequent exposure to autoradiographic film.
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4

Protein Immunodetection by Western Blot

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Cells were lysed with loading buffer (50 mM Tris pH=6.8, 2% SDS and 10% glycerol) and denatured by boiling. Protein concentration was then determined by the BCA assay (Thermo Fisher) and systematically normalized before SDS-PAGE. Following the transfer of proteins to nitrocellulose (Thermo Fisher), immunodetection was performed using the indicated primary and peroxidase-coupled secondary antibodies. Proteins were visualized by enhanced chemiluminescence (ECL, Thermo Fisher). Antibodies used include anti-Actin (Santa Cruz; sc-1616; RRID: AB_630836) and anti-GSDME (Abcam; ab216191; RRID: AB_2737000).
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5

Quantification of Lung Protein Profiles

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Pulverized mouse lungs were homogenized in RIPA buffer (50 mM Tris HCl, pH 7.4, 1% Triton X-100, 0.2% SDS, 1 mM EDTA) supplemented with an EDTA-free protease inhibitor (Roche) and protein extracts were quantified. Western blotting was performed using 15% SDS-polyacrylamide gels and membranes were probed with antibodies raised against β -actin (Santa Cruz, sc1616, 1:1000) and RELMα (Abcam, ab39626, 1:1000). Blots were incubated with horseradish peroxidase-linked secondary antibodies and immunoreactivity was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific) and visualized with a digital luminescent image analyzer (Image Quant LAS4000 Mini; GE).
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6

Mouse Brain Protein Extraction and Analysis

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Protein was extracted from homogenised adult mouse brain (~100 mg brain; 350 μl RIPA buffer containing protease inhibitor cocktail (Roche Diagnostics, Indiana, US)) by centrifugation. Protein yield was determined by Bradford assay (Bio-Rad Laboratories, Hercules, US; bovine serum albumin (BSA) standard). Protein (80 μg) was subjected to gel electrophoresis utilising Criterion XT 1.0 mm gels (Bio-Rad) and transferred to nitrocellulose membrane (Hybond-C Extra 0.45 μm; Amersham Biosciences, Rydalmere, Aus). Membranes blocked in 3% BSA-TBS/T (Tris buffered saline (TBS)/0.1% Tween-20 (T)) overnight (4 °C) were probed for SGTA using ‘full length SGTA’ (ProteinTech), ‘C-terminal SGTA’ (Aviva BioSciences, San Diego, US) or ‘N-terminal SGTA’ (Santa Cruz) antibodies diluted at 1:1,000 (full, C-terminal) or 1:500 (N-terminal) in 1% BSA-TBS/T (60 min, room temperature). Immunoreactivity was detected using polyclonal goat anti-rabbit (1:1,000) or rabbit anti-mouse IgG/HRP (1:500) antibodies (Dako) followed by chemiluminescent visualisation (Super Signal West Dura Extended Duration, Thermo Fisher Scientific, Scoresby, Aus) and ChemiDoc MP imaging (Bio-Rad). Reference proteins used were actin (Santa Cruz Biotechnology, sc-1616, 1:2,000) or Histone H-3 (Abcam, ab7766, 1:500).
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