Figure 1.

Rps3 is a novel Lxn binding protein. (a) Experimental scheme for isolation of Lxn binding protein. Full-length mouse Lxn was fused in-frame to an N-terminal tandem affinity purification (TAP) tag and sub-cloned into Sfβ9.1-GFP retroviral vector to create the TAP-Lxn vector. A plasmid carrying the TAP tag was used as a control (TAP). FDC-P1 cells were transfected with the TAP-Lxn or TAP retroviral supernatant, and GFP+ cells were sorted by flow cytometry to establish GFP+ stable cell line. Whole-cell lysates from TAP-Lxn or TAP FDC-P1 cells were purified with the InterPlay Mammalian TAP purification kit and the differentially expressed bands were subjected to mass spectrometry. (b) Detection of TAP-Lxn or TAP protein expression in stable GFP+FDC-P1 by antibodies against TAP tag (left panel) and Lxn (right panel), respectively. (c) Proteins differentially present in FDC-P1 cells transfected with TAP-Lxn or TAP vectors. (d) Rps3 protein is expressed in FDC-P1 cells. Western blot was performed with anti-Rps3 antibody. (e) Co-immunoprecipitation confirms the binding of Lxn and Rps3. Whole-cell lysate from FDC-P1 cells was incubated with Rps3 antibody or IgG control, and the precipitated proteins were probed with anti-Lxn antibody in western blot (WB:Lxn) (top panel). The binding was confirmed by the reciprocal way in which the protein complex was precipitated by Lxn antibody and then western blotted with Rps3 antibody (WB: Rps3) (bottom panel). (f) Lxn and Rps3 are colocalized in the cytoplasm of FDC-P1 cells. FDC-P1 cells were fixed and stained with immunofluorescent probes for Lxn and Rps3 proteins. Lxn was detected by polyclonal antibody and rhodamine-conjugated goat-anti-chicken secondary antibody; Rps3 was detected by polyclonal antibody and FITC-conjugated goat anti-rabbit secondary antibody. Scale bar represents 10 μm