Figure 2

(A) Schematic diagram of Cish constructs used in our experiments. (B) 293 T cells were transfected with Tagged plasmids expressing PLC-γ1-YFP, Ubiquitin-HA, and Cish-FLAG constructs (WT, SH2* and D/BC*) where indicated, in the presence of the proteasome inhibitor, MG-132. PLC-γ1-YFP (GFP antibody) was immunoprecipitated and blotted for HA and YFP. Whole-cell lysates were blotted for Cish and Cish and YFP PLC-γ1. Numbers below the band indicate PLCγ1 ubiquitin quantification. A value of 1 was arbitrarily set for PLCγ1 ubiquitination in the presence of the Cish WT construct. (C) Same as in (B) with no proteasome inhibitor, MG-132. Cish (Flag M2) was immunoprecipitated and blotted for YFP (PLC-γ1) and Cish. Whole-cell lysates were blotted for YFP. Numbers below the band indicate PLCγ1 binding to Cish quantification, a value of 1 was arbitrarily set for PLCγ1 binding in the presence of the Cish WT construct. (D) Immunoprecipitation of FLAG-tagged Cish and immunoblotting of PLC-γ1 and Cish, in indicated transduced CD8⁺ T cells with and without CD3 stimulation (5 min). Whole lysates were blotted for PLC-γ1 and Flag M2.