After stimulation, cells were washed with HBSS and then scraped in lysis buffer (1 % Triton X-100, 1X mini complete, 1 mM PMSF, 2 mM sodium orthovanadate, 20 mM sodium pyrophosphate, and 50 mM sodium fluoride). Lysates were incubated on ice for 10 min, frozen at −80 °C, thawed, sonicated, and centrifuged at 10,000 × g for 5 min at 4 °C. Protein concentrations for the Triton X-100-soluble lysates were quantified using a DC Protein Assay (Bio-Rad, Montreal, Quebec, Canada). Equivalent amounts of protein were separated by SDS-PAGE, and proteins were then transferred to a nitrocellulose membrane. Membranes were blocked with 5 % skim milk for 1 h and probed with either 1 μg/mL of the IRF7 antibody or a 1:1000 dilution of the viperin antibody overnight at 4 °C. Membranes were washed and then incubated for 1 h with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-mouse Ig antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Proteins were visualized with a Pierce ECL substrate reagent (Thermo Scientific, Rockford, IL). Equal loading was further determined probing with an antibody against GAPDH.
Pierce ecl substrate reagent
Manufactured by Thermo Fisher Scientific
The Pierce ECL substrate reagent is a chemiluminescent substrate used for the detection of horseradish peroxidase (HRP) in Western blotting applications. The reagent produces a luminescent signal upon reaction with HRP, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay, by Bio-Rad (1 mentions)
Skim milk powder, by Fujifilm (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Restore stripping buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc machine, by Bio-Rad (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
2 protocols using pierce ecl substrate reagent
1
Immunoblotting for IRF7 and Viperin
2
Western Blot Analysis of Puromycin Incorporation
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Western blotting was conducted as previously reported(Rashad et al., 2022 (link); Rashad et al., 2020a (link)). Briefly, for the analysis of puromycin incorporation assay, cells were incubated in a complete growth medium with 10 μg/ml puromycin then homogenized in T-PER™ tissue protein extraction reagent (Thermo Fisher Scientific, Cat# 78510) with Triton(R) X-100 (Nacalai Tesque, Cat# 35501-15) and cOmplete™ protease inhibitor cocktail (Roche, Cat# 4693116001). Proteins were isolated from the supernatant, quantified using the Brachidonic-acid assay kit (BCA) (Thermo Fisher Scientific, Cat# 23227), separated on Mini-PROTEAN TGX gel (Bio-Rad, Cat# 4561096), and transferred to Polyvinylidene difluoride membrane (PVDF) (Bio-Rad, Cat# 1704156). Membranes were blocked in 5% Skim milk powder (Fujifilm, Cat# 190-12865) with Phosphate buffered saline with tween® (PBS-T) (Takara, Cat# T9183). The primary antibody was incubated overnight at 4°C, followed by room temperature incubation with the secondary antibody (IgG detector solution v2). Signal detection was performed using Pierce ECL substrate reagent (Thermo Fisher Scientific, Cat# 32106) on a ChemiDoc machine (Bio-Rad). To normalize protein expression, membranes were stripped with Restore™ stripping buffer (Thermo Fisher Scientific, Cat# 21059) and reprobed with Anti-beta actin antibody.
List of antibodies used:
List of antibodies used:
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