CKIδ is dispensable for IS formation and early TCR signaling. (A) Conjugates of control (EV) or CKIδ-depleted (shdelta) Jurkat and SEE-pulsed Raji cells are stained with anti–LFA-1 antibody (top), phalloidin (F-actin; middle), or anti-CD3 antibody (bottom). Centrosomes are stained with anti-CDK5RAP2 antibody. Raji cells are in blue in merge. (B) Cytoplasmic cell extracts of control (EV) or CKIδ-depleted (shdelta) Jurkat cells were lysed at different time points after conjugate formation with SEE-pulsed Raji cells and were immunoblotted with the indicated antibodies. WB, Western blotting. (C) Conjugates between control (EV; left) or CKIδ-depleted (shdelta; right) Jurkat and SEE-pulsed Raji cells are stained with anti-DIC (green in merge) and anti-CDK5RAP2 (red in merge) antibodies. Arrowheads highlight DIC accumulation at the IS. (D and E) Cytoplasmic cell extracts (CCE) were prepared from control (EV) and CKIδ-depleted (shdelta; D) or DMSO- and D4476-treated (E) Jurkat cells that were unconjugated (−) or conjugated (+) to anti-CD3 antibody beads. Immunoprecipitations (ip) were performed with random IgG (ip con) or anti-p150glued (ip p150glued) antibodies. Bars: 10 µm; (en face) 5 µm.
