Modulation of hIAN1 protein level during T- and B-lymphocyte activation.
(A) The hIAN1, hIAN2, and hIAN7 cDNAs were in vitro transcribed/translated as described in “Materials and methods,” and the 35S-labeled protein products were run on a 10% SDS-PAGE. The same in vitro–translated products together with total protein extracts of Jurkat and Jurkat-ΔCOOH were run on a 10% SDS-PAGE, blotted, and revealed by the anti-hIAN1 antiserum. (B) T lymphocytes were purified from human peripheral blood and activated with anti-CD28 and anti-CD3. Before activation (lane 1), and 30 minutes (lane 2), 5 hours (lane 3), 1 day (lane 4), 4 days (lane 5), and 6 days (lane 6) after activation, total RNA was extracted from half of the culture and protein extract was performed on the other half. Semiquantitative RT-PCR analysis was performed in the linear range of amplification, and Western blot analysis was performed by means of the anti-hIAN1 antiserum. The loading control for the Western blot was obtained with the use of an anti-ZAP70 antibody.25 (C) B lymphocytes were purified from human PBMCs and activated with 10 mg/mL IL-4 and irradiated (75 Gy) murine L cells that expressed the human CD40 ligand. RNAs and proteins were studied before activation (lane 1) and 1 day (lane 2), 2 days (lane 3), 3 days (lane 4), and 4 days (lane 5) after activation as described in panel B; the loading control for the Western blot was an anti–β-actin antibody.
