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. Author manuscript; available in PMC: 2013 Aug 15.
Published in final edited form as: J Immunol. 2012 Jul 13;189(4):1689–1698. doi: 10.4049/jimmunol.1101484

FIGURE 6.

FIGURE 6

Potential roles for ROCK-mediated CD1d phosphorylation and RhoGTPases in CD1d-mediated Ag presentation. A, Amino acid sequences of the intracellular, carboxy-terminal tails of human CD1b, CD1c, CD1d and mouse CD1d1. B, ROCK1 and ROCK2 kinase were used in in vitro kinase assays with the indicated CD1 peptides. CREB and PLK peptides served as positive controls. Incorporation of radioactive phosphate into the peptides was measured by scintillation counting and presented in cpm. This result is representative of two independent experiments. C, HEK293-hCD1d cells in which T329 and S330 (part of the ROCK phosphorylation motif) have been mutated to alanines were transfected with ROCK1-specific or control (NC) shRNA, and used in a co-culture assay with human NKT cells. IL-4 and GM-CSF production by human NKT cells was measured by ELISA; *, P = 0.0125 for IL-4 and P = 0.0306 for GM-CSF. This result is representative of three independent experiments. D, LMTK-CD1d1 cells were treated +/− the Rho-specific inhibitor C3 toxin and co-cultured with the representative N37-1A12 NKT cell hybridoma. NKT cell production of IL-2 was measured by ELISA; *, P = 0.0001. This result is representative of two independent experiments.