Anti xor

XOR and CDK5 were precipitated from 300μg of protein lysates pre-cleared with 25μl of protein A/G agarose beads (50% slurry) at 4°C for 3 hours. Proteins were immunoprecipitated by addition of the respective primary antibody, anti-CDK5 (Santa Cruz, Santa Cruz, CA), anti-XOR (Santa Cruz, Santa Cruz, CA,), or a species control IgG and incubated overnight at 4°C. Immune complexes were captured with protein A/G agarose beads slurry and washed three times in lysis buffer or kinase activity buffer. One-fifth of the immunoprecipitation reaction was subjected to Western blot analysis, or for CDK5 activity measurements.

Cell lysates were generated by homogenization in Cell Lysis Buffer (Cell Signaling, Boston, MA) supplemented with 1x protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO, P8340) and sonicated, twice using a Fischer Scientific sonicator FB50, then cleared by centrifugation. 40μg of protein per sample were resolved by electrophoresis on 12% tris-glycine gels from Life Technologies (Carlsbad, CA) and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA). Specific proteins were detected with the following antibodies, anti-XOR (Santa Cruz, Santa Cruz, CA, #sc-22006), anti-GAPDH-HRP (Cell Signaling, Boston, MA, #3683), anti-Cdk5 (Santa Cruz, Santa Cruz, CA, #sc-6247), anti-p35 C-19 (Santa Cruz, Santa Cruz, CA, #sc-820), anti-p35 N-20 (Santa Cruz, Santa Cruz, CA, #sc-821), anti-HA tag (Santa Cruz, Santa Cruz, CA, #sc-57592), anti-histone (Cell Signaling, Boston, MA, #2935), anti-phospho-H1 (Cell Signaling, Boston, MA, #2577), anti-myc antibody (Cell Signaling, Boston, MA, #2272), and anti-phospho-threonine (Cell Signaling, Boston, MA, #9391) where indicated. Immune complexes were detected using chemiluminescence (ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA).

Cell lysates were made using RIPA buffer. Equal amounts of protein (40 μg) were electrophoresed on SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). Membranes were incubated overnight at 4°C with primary antibody. Antibodies used for western blotting were as follows: anti-XOR (#sc-398548, Santa Cruz, CA), anti-IFNGR1 (#ab134070, Abcam, Cambridge, MA), anti-CBP (#sc-7300 X, Santa Cruz, CA), anti-STAT1 (#14994, Cell Signaling Technology (CST), Danvers, MA), anti-pSTAT1 (#9167, CST, Danvers, MA), anti-STAT3 (#9139, CST, Danvers, MA), anti-pSTAT3 (#9145, CST, Danvers, MA), anti-IRF1 (#8478, CST, Danvers, MA), and anti-β-actin (#sc-130656, Santa Cruz, CA). HRP-conjugated secondary antibody was incubated for 2 h at room temperature. Protein bands were analyzed using a Bio-Rad Gel Doc system with Image Lab software.