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. Author manuscript; available in PMC: 2012 Feb 15.
Published in final edited form as: J Immunol. 2011 Jan 17;186(4):2164–2171. doi: 10.4049/jimmunol.1003236

Fig. 5. PTB from the cytoplasm and nucleus is differentially modified and mediated by activation.

Fig. 5

(A). Cytoplasmic (lanes 1-4) and nuclear (lanes 5-8) extracts were prepared from Jurkat/D1.1 cells (lanes 1 and 5) and CD4+ T cells that were unstimulated (lanes 2 and 6) or stimulated with anti-CD3 mAb for 2 h (lanes 3 and 7) or 48 h (lanes 4 and 8). PTB expression was assessed by SDS-PAGE and immunoblotting with anti-PTB antibodies. Membranes were stripped and re-hybridized with antibodies against S6 and PARP (lower two panels) to verify the separation of cytoplasmic and nuclear fractions, respectively. (B). Two dimensional gel electrophoresis (2DGE) was carried out across a pH gradient of 6-11 using cytoplasmic (left panels) and nuclear extracts (right panels) from CD4+ T cells activated for 0, 2, 24 and 48 h with anti-CD3 mAb. Black arrows indicate the major isoforms in each fraction. (C). Cytoplasmic extract from Jurkat/D1.1 cells was either left untreated or treated with 800 U lambda phosphatase for 4 h. prior to separation by 2DGE across a pH gradient of 6-11. PTB was visualized by immunoblotting.