Hrp conjugated mouse igg

The detailed immunoblotting protocol has been described previously [21 (link)]. Pellet of PBMCs was lysed with radioimmunoprecipitation assay (RIPA) buffer, and 7 μg of the cell lysate was loaded onto sodium dodecyl sulfate-polyacrylamide gels. The blotted membrane was blocked in 5% non-fat dry milk diluted in tris-buffered saline for 1.5 h at room temperature. Primary antibodies were diluted in the blocking buffer, and the membrane was incubated with the diluted primary antibodies for 19 h at 4°C. After washing, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 30 min at room temperature. After washing, the ImageQuant LAS 4000 (GE Healthcare, Hatfield, UK) was used to obtain the images. The antibodies used are as follows: STAT1 (1:1,000, rabbit, BD transduction laboratories, San Jose, CA, USA), PY-STAT1 (1:1,000, mouse, BD transduction laboratories), GAPDH (1:1,000, rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA), HRP-conjugated rabbit immunoglobulin G (IgG) (1:5000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and HRP-conjugated mouse IgG (1:5,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA).