Visualization of PLCγ1 recruitment to signaling clusters in Jurkat E6 cells. (A) Jurkat E6 cells expressing PLCwt-YFP were seeded on stimulatory coverslips. Images obtained 0.5, 2 and 5 min into the spreading process are shown. The size bar corresponds to 5 μm. (B) Jurkat E6 cells expressing LAT-CFP and PLCwt-YFP were seeded on stimulatory coverslips. Images obtained 1.5 min into the spreading process are shown. The size bar corresponds to 5 μm. (C) Same as (B) for cells expressing Slp76-CFP and PLCwt-YFP. (D) Jurkat E6 cells expressing mutant PLCγ1-YFP conjugates as indicated were seeded on stimulatory coverslips. Images obtained 1–1.5 min into the spreading process are shown. The size bar corresponds to 5 μm. (E) Murine primary CD4⁺ T cells expressing PLCγ1-YFP conjugates as indicated were seeded on stimulatory coverslips and fixed 5 min into the spreading process. The size bar corresponds to 5 μm.

Co-precipitation of PLCγ1-YFP variants with various signaling proteins. (A) Jurkat E6 cells (1 × 10⁷) expressing PLCwt-YFP were either stimulated for 1 min with C305 or left unstimulated and lysed. PLCwt-YFP was precipitated with anti-GFP. The precipitates (three left lanes) and a sample from stimulated whole-cell lysate WCL (right lane) were resolved on SDS–PAGE and probed with anti-phosphotyrosine 4G10 antibody. NC lane represents a control experiment performed without cell lysate. All lanes were from the same gel. The three left lanes were obtained at equal exposure times. WCL lane was obtained at a shorter exposure time. HC, heavy chain of Ig; LC, light chain of Ig. The data are representative of three independent experiments. (B) Jurkat E6 cells (1 × 10⁷) expressing the indicated PLCγ1-YFP variants were either stimulated with C305 for 1 min or left unstimulated. The cells were lysed and PLCγ1-YFP conjugates were precipitated with anti-GFP. The precipitates were resolved on SDS–PAGE and probed with anti-phospho-c-Cbl-pY774 (top blot). The same membrane was stripped and probed with anti-GFP (second from top blot). In a different experiment, the precipitates were resolved on SDS–PAGE and probed consecutively with anti-phospho-Vav1-pY160, anti-Slp76 and anti-GFP (lower blots from top to bottom). NS, nonspecific band. The data are representative of at least four independent experiments. (C) The data represent densitometry of phospho-c-Cbl bands from experiments in (B) as a percentage of band intensity obtained in cells expressing PLCwt-YFP. Each bar represents an average of five independent experiments. Error bars indicate s.e. (D) Same as (C) for phospho-Vav1 bands. Each bar represents an average of four independent experiments.

Effect of Slp76 and c-Cbl deficiencies on PLCγ1 recruitment and activation. (A) Jurkat J14 cells expressing PLCwt-YFP were seeded on a stimulatory coverslip. An image obtained 1 min into the spreading process is shown. The size bar corresponds to 5 μm. (B) Jurkat J14 cells expressing c-Cbl-CFP and an indicated PLCγ1-YFP conjugate were seeded on stimulatory coverslips. Images obtained 1–1.5 min into the spreading process are shown. The size bar corresponds to 5 μm. (C) Time course of the response in cytosolic calcium concentration obtained from the indicated cell lines after stimulation with OKT3. AU, arbitrary units. (D) CD4⁺ T cells from c-Cbl KO mice expressing PLCwt-YFP were seeded on a stimulatory coverslip, fixed 5 min into the spreading process and stained with anti-pY antibody. The size bar corresponds to 5 μm.

Schematic model of PLCγ1 recruitment and activation in the LAT-nucleated signaling complex.

Effect of SH domain mutations on PLCγ1 recruitment and phosphorylation. (A) Jurkat E6 cells (1 × 10⁷) expressing the indicated PLCγ1-YFP variants were either stimulated with C305 for 1 min or left unstimulated. The cells were lysed and PLCγ1-YFP conjugates were precipitated with anti-GFP. The precipitates were resolved on SDS–PAGE and probed with anti-phospho-LAT-pY132 (upper blot). The same membrane was stripped and probed with anti-GFP (lower blot). The data are representative of three independent experiments. (B) Quantitative analysis of blots obtained in experiments represented by (A). The data represent densitometry of LAT-pY132 bands as a percentage of band intensity obtained in cells expressing PLCwt-YFP. Each bar represents an average over four independent experiments. Error bars indicate s.e. (C) Jurkat E6 cells expressing the indicated PLCγ1-YFP variants were either stimulated with OKT3 for 1 min or left unstimulated and lysed. The lysates were resolved on SDS–PAGE and probed with either anti-phospho-PLCγ1-pY783 (upper blot) or anti-PLCγ1 (lower blot). The data are representative of five independent experiments. (D) Quantitative analysis of blots obtained in experiments in (C). The data represent densitometry of PLCγ1-YFP-pY783 bands as a percentage of endogenous PLCγ1-pY783 band intensity in corresponding lanes. The results were normalized to account for the relative amounts of PLCγ1-YFP and endogenous PLCγ1 in each cell line. Each bar represents an average over five independent experiments. Error bars indicate s.e.

FRET analysis between PLCγ1-YFP and various CFP-conjugated proteins. (A) Jurkat E6 cells expressing PLCs-YFP and LAT-CFP were seeded on a stimulatory coverslip and fixed 2 min into the spreading process. FRET efficiency values are presented in a pseudo-colored scale, with black color representing saturated pixels. The average value of FRET efficiency±s.e. obtained for a given pair is shown. The size bar corresponds to 5 μm. (B) Same as (A) for cells expressing PLCs-YFP and c-Cbl-CFP. (C) Same as (A) for cells expressing PLCs-YFP and Vav1-CFP. (D) Same as (A) for cells expressing PLCs-YFP and CFP-Slp76. (E) Same as (A) for cells expressing PLCs-YFP and Slp76-CFP. (F) Same as (A) for cells expressing PLCs-SH2N^*-YFP and LAT-CFP.

Effect of Vav1 deficiency on PLCγ1 recruitment and activation. (A) Jurkat E6 cells (two left lanes) and Jurkat J.vav cells (two right lanes) were either stimulated with OKT3 for 1 min or left unstimulated and lysed. The lysates were resolved on SDS–PAGE and probed with either anti-phospho-PLCγ1-pY783 (upper blot) or anti-PLCγ1 (lower blot). The data are representative of two independent experiments. (B) Time course of the response in cytosolic calcium concentration obtained from the indicated cell lines after stimulation with OKT3. AU, arbitrary units. (C) Jurkat E6 or J.vav cells (1 × 10⁷) expressing PLCwt-YFP were either stimulated for 1 min with C305 or left unstimulated and lysed. PLCwt-YFP was precipitated with anti-GFP. The precipitates were resolved on SDS–PAGE and probed with anti-phosphotyrosine 4G10 antibody. The data are representative at least two independent experiments. (D) Jurkat J.vav cells expressing an indicated PLCγ1-YFP conjugate were seeded on stimulatory coverslips. Images obtained 1–1.5 min into the spreading process are shown. The size bar corresponds to 5 μm.