Figure 8.

Natural and recombinant chIL-9 is potentially glycosylated with a molecular weight of 25 kD. PBMCs isolated from chicken were stimulated with PMA and Ionomycin in the presence or absence of brefeldin A for 4 h. The total protein was extracted from the activated PBMCs with RIPA lysis buffer (A) and the supernatant without brefeldin A inhibitor (B) was collected for SDS-PAGE and Western Blotting. Natural chIL-9 expressed by the activated chicken PBMCs was detected inside the cells (A) and in the supernatant (B) by four chIL-9 mAbs (3H5, 4F7, 4H7, and 5B8) and rabbit anti-chIL-9 pAb (rpAb), respectively. The whole cell lysates (C) and supernatant (D) of pEGFP-chIL-9-transfected DF-1 cells treated with or without deglycosylation enzyme was recognized with anti-chIL-9 pAb and mAb 4F7, showing a reduction of molecular weight.