Grb2 is necessary for FcγR-induced SHIP-LyGDI association.

<p>THP-1 cells were stimulated for 15 minutes using CD32 antibody followed by goat anti-mouse IgG secondary antibody. Lysates were made and immunodepleted for Shc or Grb2. (A) Non-depleted, Shc-depleted and Grb2-depleted lysates from resting and activated cells were analyzed by Western blotting with anti-Shc (upper panel) and anti-Grb2 (middle panel). The same membrane was reprobed with anti-GAPDH (lower panel) to ensure equivalent protein loading. (B) SHIP was immunoprecipitated from resting and activated immunodepleted lysates and analyzed by Western blotting with LyGDI antibody (upper panel). The same membrane was reprobed with anti-SHIP to ensure equivalent protein loading (lower panel). Control IPs were done using normal mouse IgG. (C) 0.5 µg of purified GST, SHIP-GST-SH2 and SHIP-GST-PRD fusion proteins were analyzed by Western blotting with anti-GST antibody. (D) N-terminal biotinylated control peptide ‘C’ or phosphorylated peptide containing to the FcγRIIb ITIM sequence (P_ITIM) immobilized on NeutrAvidin beads were used to pulldown SHIP-GST-SH2, SHIP-GST-PRD or GST alone from solution. Bead-bound proteins were analyzed by Western blotting with GST antibody. (E) GST, SHIP-GST-SH2 or SHIP-GST-PRD were added to resting ‘R’ or FcγRIIa/IIb-stimulated ‘A’ THP-1 lysates and GST pulldowns were performed by adding glutathione agarose beads. Bead-bound proteins were analyzed by Western blotting with anti-Grb2 (upper panel). The same membrane was reprobed with anti-GST (lower panel). (F) GST, SHIP-GST-SH2 or SHIP-GST-PRD were added to resting ‘R’ or FcγRIIa/IIb-stimulated ‘A’ THP-1 lysates and LyGDI was immunoprecipiated. The immunoprecipitates were analyzed by Western blotting for SHIP (upper panel). The same membrane was reprobed with anti-LyGDI antibody (lower panel).