GST-CRKL-SH2 pull down experiment showing the effect of PMA on CBL-CRKL dissociation and CBL dephosphorylation. U937IF cell lysates were prepared and were precipitated with GST fusion protein as described in Methods. 10 μg of GST fusion protein was used for invitro pull down experiments. 50 μl of glutathione-sepharose beads (prewashed with extraction buffer) was added to each sample. GST-Crkl-SH2 fusion (lanes 2–11) protein was used to precipitate associated protein with or without PMA treatment in FcγRI stimulated cell lysate. Lane 1, precipitated with GST alone. Lane 2, no stimulation, Lane 3, 4 & 5, stimulated cell lysate (stimulated with 32.2 F(ab)2 antibody and cross linked with secondary antibody for 1, 5 and 10 min. Lane 6, 7 and 8, cells were treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations, lane 9, 10 and 11, cells were preincubated with GF109203X (2.5 μM) for 15 min. on ice and then treated with PMA (200 ng/ml) for 5 min. followed by FcγR1 stimulations. Proteins bound to GST fusion protein were resolved in 10% SDS-PAGE, blotted on nitrocellulose membrane and probed with phosphotyrosine antibody (4G10). This blot was reprobed with anti-CBL antibody. This experiment was repeated two times.
Joshi et al. BMC Immunology 2014 15:18 doi:10.1186/1471-2172-15-18