R4 and GAR domains of nucleolin are necessary for its interaction with Fas. (A) Schematic of the nucleolin domains and its known modifications: N-terminal domain region (NDR), nuclear localization signal (NLS), RNA binding domains 1-4 (R1-4), and glycine/arginine-rich domain (GAR). Glycosylation sites are represented by Y, phosphorylation sites by yellow circles, protein-binding sites by stars, and 3 defined proteolytic cleavage sites (of a potential 18 putative sites) by a blue lightning bolt. (B) Domain deletions were created by using the Stratagene Quick Change II XL mutagenesis kit using C-terminal DDK/myc-tagged PCMV-ENTRY construct of full-length nucleolin (Origene) as a template. (C) 293T HEK cells were transfected with the indicated nucleolin domain deletion mutants and lysed for IP/IB analysis. Whole-cell lysates were subjected to Fas IP with agarose conjugated with B-10 anti-Fas antibody. Proteins were separated and immunoblotted for detection of coprecipitated domain mutants with an anti-DDK-HRP antibody. A mixture of all domain mutants was precipitated with mouse IgG and protein G agarose as a negative control. Whole-cell lysate samples prior to IP were immunoblotted with anti-DDK-HRP to reveal expression levels of the transfected constructs. Representative data from 3 different experiments are shown. (D) A chimeric Fc:Fas (Fc:extracellular domain of Fas) was incubated with varying concentrations of recombinant nucleolin-GST for 1.5 hour at 4°C. Fc:Fas was immunoprecipitated with protein A overnight and precipitates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. IB revealed nucleolin-GST present in Fas-precipitated complexes in a dose-dependent manner. Mouse IgG-1 was used as a control for Fc fragment binding.