Research Paper Volume 13, Issue 7 pp 10490—10516

Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells

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.

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.