Figure 4.
The lamin Ig-fold motif binds PCNA. (A) Bacterially expressed and purified PCNA was separated by SDS-PAGE and stained with Coomassie blue (PCNA, molecular weight markers on the left). We always observe that the expressed PCNA appeared as one major and one minor band, both of which immunoblotted with anti-PCNA (see E). (B) LA, lamin A tail (LAT), LB1 tail (LB1T), LC tail (LCT), X. laevis LB3 tail (LB3T), and the Ran-binding domain of importin β (Imp βR) were expressed, purified, bound to protein S–agarose beads, and then mixed with purified PCNA. + lanes show experiments where PCNA was added to S-tagged proteins bound to protein S–agarose beads, whereas − lanes show omitted PCNA. The proteins bound to the S–agarose beads were eluted in SDS sample buffer and separated by SDS-PAGE. Gels were stained with Coomassie blue. The results show that LA, LAT, LB1T, LCT, and LB3T bind to PCNA (arrow). (C) The area identified by the arrow (∼37 kD) in Fig. 4 B was enhanced to more easily identify PCNA bound to LB3T. (D) S-tagged LA-Ig was bound to protein S–agarose beads and incubated with PCNA. The bound proteins were separated by SDS-PAGE, transferred to nitrocellulose, and stained with India ink. The location of LA-Ig and PCNA are shown. (E) After immunoblotting, the nitrocellulose blot seen in F (left) was briefly stained with India ink to show that equal amounts of Imp βR (∼50 kD), LA-Ig, and LA Ig-fold R453W (LA-Ig RW; ∼25 kD) were bound to protein S–agarose beads. (F, left) Purified Imp βR, the LA-Ig, and LA-Ig RW were bound to protein S–agarose beads and mixed with purified PCNA. The bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. The separated proteins were transferred to nitrocellulose and immunoblotted with antibodies directed against PCNA. S-tagged Imp βR was used as a negative control. It was found that the amounts of PCNA bound both to protein S–agarose beads alone (not depicted) and to Imp βR bound to protein S–agarose beads were equivalent (Imp βR +). Therefore, this amount of PCNA binding was considered background. Densitometric analysis showed that after subtracting this background, PCNA binding to LA-Ig RW bound ∼28% less PCNA than wild-type LA-Ig. (F, right) Densitometric analysis of the bound PCNA by immunoblotting seen in F (left). Error bars indicate standard deviation of the mean for six experiments.