Figure 4.
Precipitation of the 36-kDa band from activated cell lysates with a GST-Grb2 fusion protein. CTL (3 × 106) from the HLA A2-restricted line recognizing HIV-1 p17-8 Gag were activated by presenting with 3 × 105 APC pulsed with 50 μM agonist peptide (SLYNTVATL). Either a GST-Grb2 fusion protein or GST protein was added to the cell lysate, and fusion protein complexes then were precipitated as described in the text. Proteins in lysate cleared by GST-Grb2 (lane 3), in the GST-Grb2 precipitate (lane 4), and in the GST precipitate (lane 5) were separated by SDS/PAGE and immunoblotted with anti-phosphotyrosine mAb. Untreated detergent-soluble fractions from lysates of resting (lane 1) and activated (lane 2) CTL were run as controls. The band of 38 kDa that weakly bound to the GST-Grb2 fusion protein was identified as phospho-p38 mitogen-activated protein kinase (MAPK) by reprobing the blot with anti-p38 MAPK Ab (data not shown).