Mouse anti mx1 monoclonal antibody

The splenic samples were homogenized in RIPA Lysis Buffer (Biosharp, BL504A) with protease inhibitor. The protein concentration was calculated using a BCA Protein Assay kit (Tiangen Biotech), and total proteins were separated by electrophoresis on 12% SDS-PAGE gels. Total proteins were transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then the membranes were blocked with 5% skimmed milk powder. The membranes were incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a rabbit anti-OAS1 polyclonal antibody (Abcam, ab86343, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), and a mouse anti-CXCL10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), respectively. After washing, the membranes were incubated with goat anti-mouse IgG-HRP (Biosharp, BL001A) or rabbit anti-goat IgG-HRP (Biosharp, BL004A) with 1:10 000 dilution, and the signals were detected using an ECL western blotting detection reagent (Tiangen Biotech). The immunospecific bands were quantified using Quantity One V452 (Bio-Rad Laboratories, Hercules, CA, USA) with GAPDH as an internal control protein. GAPDH was detected using an anti-GAPDH antibody (Santa Cruz Biotechnology, Inc., sc-20357, 1:1000).

Proteins in the thymic samples were extracted using RIPA lysis buffer (Biosharp, BL504A), and protein concentration was measured with a BCA Protein Assay kit (Tiangen Biotech). Total proteins (10 μg/lane) were separated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. The membrane was blocked in 5% skimmed milk powder, and then incubated with a goat anti-STAT1 polyclonal antibody (Abcam, Cambridge, UK, ab230428, 1:1000), a mouse anti-Mx1 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-166412, 1:1000), a mouse anti-IP-10 monoclonal antibody (Santa Cruz Biotechnology, sc-374092, 1:1000), and a mouse anti-UBE1L monoclonal antibody (Santa Cruz Biotechnology, sc-390097, 1:1000), respectively. Membranes were washed three times, and incubated with goat anti-mouse IgG-HRP (Biosharp, BL001A) or rabbit anti-goat IgG-HRP (Biosharp, BL004A) in a 1:2000 dilution. After washed three times, proteins detected by a pro-light HRP chemiluminescence kit (Tiangen Biotech) according to the manufacturer’s instructions. A GAPDH antibody (Santa Cruz Biotechnology, Inc., sc-20357, 1:1000) was used to monitor sample loading. The blots were quantified using Quantity One software (v450; Bio-Rad Laboratories, Inc., Hercules, CA).