Figure 3

MSP58 interacts with importin α1 and α6. A. Interaction of MSP58 with importins in a yeast two-hybrid assay. Yeast transformants with bait and prey as indicated were spotted on histidine-containing (−TULL), without histidine (−TULLH), and both histidine- and X-Gal-containing (−TULL + X-Gal) media (left) in plates. LexA-lamin served as a negative control. Yeast cotransformed were analyzed by quantitative β-Gal assays (middle). Data represent the mean ± the standard deviation of three separate experiments. A Western blot shows expression levels of different importins in yeast cells (right). B. Coimmunoprecipitation assays. Whole cell lysates from HT1080 cell lines stably expressing HA-MSP58 were subjected to immunoprecipitation (IP) experiments followed by a Western blot analysis with the indicated antibodies. IP by mouse immunoglobulin G (IgG) was used as a negative control. C. GST and fusion proteins GST-importins α1 and α6 were expressed in Escherichia coli BL21 cells and immobilized on glutathione-Sepharose. The MSP58 was obtained with a cell-free transcription and translation system in vitro and incubated with GST, GST-importin α1, or α6 proteins. Bound proteins were detected by immunoblotting with an anti-HA antibody. Coomassie blue-stained gel shows the input of GST-fusion proteins used. D. Schematic presentation of wild-type and different mutants of MSP58 tested in yeast two-hybrid assays (left). Numbers indicate the amino acid position. Yeast cotransformed with bait and prey as indicated were analyzed by quantitative β-Gal assays (right). Data represent the mean ± the standard deviation of three separate experiments. LexA-lamin served as a negative control. Immunoblotting shows the expression levels of different LexA-MSP58 fusion proteins in yeast. E. GST and GST-importin α1 or α6 proteins were immobilized on glutathione-Sepharose and incubated with cell lysates containing EGFP MSP58 1–100 or EGFP MSP58 102–300. Bound proteins were resolved by SDS–PAGE followed by Western blot analysis using anti-GFP antibodies.