M-4 cytoplasmic tail binds to ubiquitinated proteins and is modified itself by ubiquitination.

<p>(<b>A</b>) HEK-293T cells co-transfected with plasmids expressing HA-tagged ubiquitin, CD8α and each CD8β isoform or mutant proteins. After 48 hours cells were lysed with 1% BRIJ 97 and immunoprecipitated with anti-CD8β mAb, run on a polyacrylamide gel, transferred to a membrane and probed with the anti-HA antibody. Gels were reprobed with an anti-CD8β antibody (5F2). Small differences in size reflect different lengths of the isoforms. (<b>B</b>) Co-transfection of HEK-293T cells with either wild type HA-Ub or I⁴⁴A mutant of HA-Ub. The immunoprecipitation and Western blotting procedures were performed as in (A). (<b>C</b>) Determination of direct ubiquitination of CD8β isoforms. Cells expressing individual isoforms were immunoprecipitation with anti-HA mAb followed by blotting with anti-CD8β antibody. An aliquot of total cell lysate was similarly analyzed. (<b>D</b>) Effect of lysine mutants on M-4 ubiquitination. Cells expressing M-4 wild-type, single lysine (K²³⁴R, K²⁴²G) or double lysine (K²³⁴R/K²⁴²G labeled KRKG) mutants were lysed and protein analyzed as described in (C). (<b>E</b>) The JM T cells expressing CD8α and either CD8β M-4 or the double lysine mutant KRKG were incubated with the anti-CD8α (OKT8) and anti-CD3 (OKT3) mAbs for 0, 2.5 or 7 minutes at 37°C. Cells were lysed, immunoprecipitated with the anti-Ub mAb followed by Western blotting with the anti-CD8β antibody. A representative of three independent experiments is shown in each panel.