Figure 3.

Rfp2 is monoubiquitinated in cells. (A) Analysis of endogenous protein levels of Rfp2 in a panel of human cell lines. Protein lysates were prepared from neuroblastoma (SHSY5Y), neuroepithelioma (SK-N-MCIXC), Burkitt's lymphoma (Daudi), acute lymphoblastic leukemia (RS-4), keratinocyte (Rhek-1), and human diploid fibroblast (HDF) cells and subjected to Western blot analysis with Rfp2 antibodies. Nonubiquitinated Rfp2 is marked with a solid arrowhead, and asterisks indicate Rfp2 conjugated with single ubiquitin molecules. Tubulin was used as a loading control. (B) Identification of Rfp2 bands with higher molecular weight as ubiquitinated forms of the protein. GST-tagged Rfp2 and an empty vector were expressed in HEK293 cells, precipitated with Rfp2 antibodies, separated by SDS-PAGE, and visualized with Coomassie. Each Rfp2 band was excised, subjected to in-gel tryptic digestion, and analyzed by mass spectrometry. The bands analyzed are marked with numbers (1–3) and solid arrowheads. High-molecular-mass Rfp2 bands are marked with asterisks (monoubiquitinated Rfp2). Boxed text summarizes the mass spectrometric data and indicates the number of peptides identified and corresponding sequence coverage of the isolated nonubiquitinated (band 1) and monoubiquiti-nated Rfp2 protein bands (bands 2 and 3). (C) GST-Rfp2 was overexpressed together with HA-tagged ubiquitin in HEK293 cells and precipitated with glutathione beads. This was followed by Western blot analysis with HA antibodies to detect ubiquitinated Rfp2 (left) and Rfp2 antibodies to detect both unconjugated and ubiquitinated Rfp2 (panel). Molecular size markers are shown on either side in kDa.