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. 2006 Oct 2;26(23):8942–8952. doi: 10.1128/MCB.00305-06

FIG. 1.

FIG. 1.

APPL, APPL2, striatin, and SG2NA coimmunoprecipitate with GIPC. (A) Identification of GIPC-associated proteins by mass spectrometry. Anti-GIPC and control immunoprecipitates from PC12(615) cells were resolved on SDS-PAGE and silver stained. Protein bands present in the GIPC IP but not the preimmune (Pre) IP were identified by mass spectrometry as striatin, SG2NA, APPL, APPL2, and GIPC (arrows). (B) Confirmation of the presence of APPL, striatin, and SG2NA in the anti-GIPC IPs by immunoblotting. (C) GIPC binds directly to APPL. GST-GIPC or GST (1.5 μg) was incubated with purified HA/His6-APPL (2 μg) and glutathione beads. Bound proteins were separated by SDS-PAGE and stained with Coomassie blue. APPL was pulled down by GST-GIPC (lane 5) but not by GST alone (lane 4). Lanes 1, 2, and 3 are, respectively, input GST, input GST-GIPC, and protein standards (200, 116, 97, 66, 45, 30, and 21 kDa). (Bottom) Immunoblotting confirmed that APPL was pulled down by GST-GIPC (lane 5) but not by GST alone (lane 4). (D) The C-terminal PDZ-binding motif of APPL is required for binding to GIPC. GST-GIPC pulled down APPL (lane 2) but not APPLΔC (lane 4) from cell lysates, while GST did not pull down either protein (lanes 1 and 3). Beads with bound GST-GIPC or GST (3 μg) were incubated with 1 mg cell lysate containing APPL or APPLΔC. Bound proteins were stained with Coomassie. (Bottom) Immunoblots of inputs (20 μg lysate). FL, full length. (E) GIPC(LG>AE) binds to TrkA but not to APPL. FLAG-tagged GIPC and GIPC(LG>AE) were expressed in PC12(615) cells and immunoprecipitated with anti-FLAG IgG. The precipitates were immunoblotted for TrkA, APPL, and FLAG.