Figure 4

Rlk is associated with Lipid RAFTs: RAFT association does not require tyrosine phosphorylation, but does require the Cysteine-string. (A) Jurkat-Tag cells were electroporated with Rlk-GFP and 24 hours later were either left unstimulated or stimulated with anti-CD3e. Cells were lysed in 1% Triton X-100 and lysates fractionated over a 40%-30%-5% sucrose step gradient for 16 hr at 200,000 g. Collected fractions were separated by SDS-PAGE and analyzed for presence of Fyn, Rlk and PLC-γ1. RAFTs are included in fraction 3 or fractions 3-4. (B) Jurkat-Tag cells were electroporated with either Rlk-GFPKI, Rlk-GFPY420F or Rlk-GFPCM and treated as in (A). Only fractions 3,4 and 10 are shown. For Rlk-GFPCM, a longer exposure is shown to demonstrate the residual RAFT association. (C) Jurkat-Tag cells were electroporated with Rlk-GFP, treated with Wortmannin or carrier (DMSO) and fractionated as above.