Fig. 4.

MO-mediated ADAP knockdown abolishes MTOC polarization in Jurkat cells. Jurkat cells were electroporated with either an antisense MO directed against ADAP (ADAP MO) or a standard control MO 24 h before experiment. Normal (untreated), ADAP MO, and control MO Jurkat cells were used in the preparation of Jurkat (J)–Raji (R) cell pairs as described. (a–d) Normal Jurkat–Raji pairs shown in bright field (a) were immunostained for α-tubulin (b) and ADAP (mouse anti-fyb, clone 5) (c) with the red–green overlay shown in d. The results show typical ADAP clustering and MTOC polarization. (e–h) ADAP MO-treated Jurkat–Raji pairs prepared as in a–d show a loss of ADAP from the synapse and failure of MTOC polarization. (i–j) Control MO prepared as in a–d show normal ADAP clustering and normal MTOC polarization. (k) A bar graph summarizing polarization counts demonstrates a dramatic reduction in MTOC polarization in the ADAP MO Jurkat cells compared with normal and control MO Jurkat cell preparations. Note that background polarization levels obtained by treating Jurkat cells with the Srk kinase inhibitor PP2 are 25%. Data are representative of three independent experiments. Normal, n = 50 cell pairs; control MO, n = 50 cell pairs; ADAP MO, n = 50 cell pairs. Error bars represent ± SD. (Scale bars: 5 μm.)