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. 2011 Aug 16;30(20):4274–4286. doi: 10.1038/emboj.2011.281

Figure 1.

Figure 1

PIKE associates with GRIP1. (A) Y2H screening using PIKE-L GTPase domain as the bait. (B) Both PIKE-A and PIKE-L interact with GRIP1. HEK293 cells were co-transfected with myc–GRIP1 and various GFP-tagged PIKE-L or PIKE-A constructs. PIKE proteins were immunoprecipitated and the associated GRIP1 was detected (first panel). The expression of GFP proteins (second and third panels) and myc–GRIP1 was also examined (fourth panel). (C) PIKE-L associates with GRIP1 in neurons. Immunoprecipitation using various antibodies was performed as indicated in cultured cortical neurons (21 DIV) and the associated proteins were detected using specific antibody (top panel). Total GRIP1 (middle panel) was detected using antibody against GRIP1. Antibody against the C-terminus of PIKE-L (PIKE (C)) was used to determine the expression of PIKE-L (bottom panel). (D) PIKE-L associates with GRIP1 in rat brain. Immunoprecipitation using indicated antibody was performed in whole brain lysates and the associated PIKE-L was detected by antibody against the N-terminus of PIKE-L (PIKE (N)). (E) Co-localization of PIKE-L and GRIP1 in neurons. Immunofluorescent staining was performed on hippocampal neurons (21 DIV) using anti-PIKE-L C-terminus antibody (PIKE (C)) (green) and anti-GRIP1 antibody (red). Scale bar represents 20 μm. (F) Schematic representation of various GRIP1 deletion truncates used in the in vitro binding assay. (G) Mapping of PIKE-L interaction domain in GRIP1. Various deletion truncates of GRIP1 tagged with bacterial GST were purified and incubated with cell lysates from HEK293 cells expressing HA–PIKE-L. The GST proteins were pulled down and the associated PIKE-L was detected (upper panel). The expression of GST-tagged GRIP1 truncates (asterisked) was also examined (lower panel). (H) Schematic representation of various PIKE-L deletion truncates used in the in vitro binding assay. (I) Mapping of GRIP1 interaction domain in PIKE-L. Various deletion mutants of PIKE-L were expressed and purified from bacteria and incubated with cell lysates from HEK293 cells expressing myc–GRIP1. The GST proteins were pulled down and the associated PIKE-L was detected (upper panel). The expression of GST-tagged PIKE-L truncates (asterisked) was also examined (lower panel).