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. 2005 Mar;25(6):2227–2241. doi: 10.1128/MCB.25.6.2227-2241.2005

FIG. 2.

FIG. 2.

G3BP decreases Csk function and sequesters Csk away from lipid rafts. (A) The top panel shows an anti-Lck-phospho-Y505 immunoblot of lysates from Jurkat T cells transfected with GFP (lane 1) or GFP-G3BP (lane 2) and sorted for green fluorescence. The middle panel shows anti-Lck immunoblotting of the same filter to verify equal loading. The bottom panel shows anti-G3BP immunoblots of the same lysates. Note that endogenous G3BP is present in both lanes, but the transfected GFP-G3BP is only seen in lane 2. (B) The upper panel shows anti-Csk immunoblot of C305-treated (lanes 1 to 3) or resting (lanes 4 to 6) control Jurkat T cells fractionated into buoyant detergent-insoluble (lipid rafts), detergent-soluble fractions (soluble), or detergent-free soluble (cytosol) fractions. The lower panel shows anti-Csk immunoblot of a similarly fractionation of G3BP-transfected cells. Equal amounts of protein were loaded in each lane between the upper and lower panels. (C) The top panel shows anti-G3BP immunoblots of lipid raft fractionation of resting Jurkat T cells. Lipid rafts are in fractions 1 and 2. The second to fifth panels show the same experiment with cells stimulated through the TCR for 5, 10, 30, and 60 min. The sixth and seventh panels show immunoblots with anti-PAG, anti-Lck, and anti-Csk antibodies of the same fractions as in the top panel. The seventh panel is a longer exposure (without anti-Lck) to show Csk better. The bottom panel shows an anti-LAT blot of the same fractions as a lipid raft marker. (D) The upper panel shows an anti-Lck-phospho-Y506 immunoblot of lysates from Jurkat T cells transfected with control siRNA (lane 1) or G3BP siRNA (lane 2) together with a fluorescent RNA oligonucleotide and sorted for green fluorescence. The lower panel shows anti-G3BP immunoblots of the same lysates.