Research Paper Volume 13, Issue 7 pp 10490—10516

Figure 4. The coil region of RAP1 and the potential phosphorylation of residues in that domain are likely critical for the SUN1 interaction. (A) Schematic representations of the N-terminal HA-tagged RAP1 constructs and the N-terminal EGFP-tagged SUN1 constructs. TM, transmembrane. (B) U2OS cells were transfected with HA-tagged RAP1 together with either EGFP or EGFP-tagged SUN1 N205. Forty-eight hours posttransfection, the cells were harvested for use in immunoprecipitation assays. EGFP-SUN1 N205 was immunoprecipitated with anti-GFP beads. Input and immunoprecipitated proteins (IPs) were analyzed by immunoblotting with anti-GFP and anti-HA antibodies. Asterisk (*), nonspecific band. (C) A schematic representation of the eight potential phosphorylation sites in the coil domain of RAP1. (D) U2OS cells were transfected with HA-tagged RAP1 WT, nonphosphorylatable (8FA), or phospho-mimetic (8DE) mutant together with EGFP-tagged SUN1 N205. Forty-eight hours posttransfection, the cells were harvested for use in immunoprecipitation assays. Input and immunoprecipitated proteins (IPs) were analyzed by immunoblotting with anti-GFP and anti-HA antibodies. Asterisk (*), nonspecific band.