Both, Flag-tagged M53/p38 and HA-tagged M50/p35 can efficiently pull

In order to further improve the efficiency of the pull down assay to identify cellular interaction partners of M50/p35 and M53/p38 additional pull down systems were established: The HA-tag pull down and the Flag-tag pull down.

To test the specificity of HA-pull down 293 cells were either mock transfected, transfected with pM53, expressing wt M53, transfected with pHAM50, expressing HA-tagged M50 or co-transfected with pHAM50 and pM53. As controls for protein load- and expression total cell lysates were separated by SDS-PAGE and subjected to Western-blot analysis using M53- and M50-specific antisera at dilutions of 1:2000 (Fig. 33A, T, upper- and middle panel).

The specific M53-band at 38 kDa became detectable for total cell lysates after isolated expression of M53/p38 and after co-expression of wt M53 and HA-tagged M50 (Fig. 33A, upper panel, T, lanes 2, 4). The specific M50-band at 35 kDa was present in total cell lysates after isolated expression of HAM50 and

D. Results after co-expression of wt M53 and HA-tagged M50 (Fig. 33A, middle panel, T, lanes 3, 4).

A Figure 33A. HA-pull down analysis.

293 cells were mock transfected, transfected with HAM50, transfected with wt M53 or co-transfected with HAM50 and wt M53. Total cell lysates were analyzed by SDS-PAGE and Western blot using specific M53/p38 antiserum (upper panel, M53, T) or specific M50/p35 antiserum (middle panel, M50, T). Proteins complexed with HAM50 were precipitated with HA-matrix sepharose beads and eluted by specific IP loading buffer. Next, proteins were separated by SDS-PAGE and signals for M53/p38 were visualized by Western blot using specific antiserum against M53/p38 (lower panel, M53, B).

In the HA- tag pull down assay only proteins bound to M50 should be recovered by HA-matrix sepharose beads from lysates of 293 cells transfected with pHAM50 or lysates of co-expressed pHAM50 and pM53. Bound proteins were eluted from the HA-matrix by SDS-sample buffer, separated by SDS-PAGE and subjected to Western-blot analysis using M53-specific antisera. The M53/p38 specific band at 38 kDa was clearly detectable only after co-expression of M53 with HA-tagged M50, but was missing for the other controls (Fig. 33A, B, lanes 5-8). No signals for unspecific bound proteins were detectable, indicating the high specificity of this assay.

Next, the Flag-tag pull down assay was tested. To test the specificity of the Flag-tag pull down 293 cells were either mock transfected, transfected with pFlagM53, expressing Flag-tagged M53 or co-transfected with pFlagM53 and pM50. To control protein load- and expression the total cell lysates were separated by SDS-PAGE and subjected to Western-blot analysis using M50-specific antisera at a dilution of 1:2000. Here, the M50-specific M50-band at 35 kDa and signals for M50-processing products became detectable in total cell lysates

D. Results after co-expression of wt M50 and Flag-tagged M53 (Fig. 33B, upper panel, T, lane 3). As a further control for protein load- and expression total cell lysates were separated by SDS-PAGE and subjected to Western-blot analysis using M53-specific antisera at a dilution of 1:2000. The specific M53-band at 38 kDa was seen only in total cell lysates after isolated expression of Flag-tagged M53 or after co-expression of wt M50 and Flag-tagged M53 (Fig. 33B, middle panel, T, lanes 2, 3).

B Figure 33B. Flag-tag pull down

analysis. 293 cells were mock transfected, transfected with FlagM53, or co-transfected with FlagM53 and wt M50. Total cell lysates were analyzed by SDS-PAGE and Western blot using specific M50/p35 antiserum (upper panel, M50, T) or specific M53/p38 antiserum (middle panel, M53, T).

Proteins complexed with FlagM53 were precipitated with Flag-matrix sepharose beads and eluted by specific IP loading buffer. Next, proteins were separated by SDS-PAGE and signals for M50/p35 were visualized by Western blot using specific antiserum against M50/p35 (lower panel, M50, B).

Flag-tag pull down should only reveal proteins bound to Flag-tagged M53.

Bound proteins were eluted from the Flag-matrix by SDS-sample buffer, separated by SDS-PAGE and subjected to Western-blot analysis using M50-specific antisera. M50/p35 and processing products were only detectable after co-expression with Flag-tagged M53/p38, indicating the high specificity of the assay (Fig. 33B, lower panel, B, lane 3).

Next, the interaction of LBR with M50/p35 or M53/p38 alone or with the M50/p35-M53/p38 complex was re-analyzed. For the HA-tag pull down 293 cells were mock transfected, transfected with pHAM50 or co-transfected with

D. Results pHAM50 and pM53. For Flag-tag pull down 293 cells were mock transfected, transfected with pFlagM53 or co-transfected with pM50 and pFlagM53. As control for protein load- and expression total cell lysates were separated by SDS-PAGE and subjected to Western-blot analysis using LBR- specific antibody at a dilution of 1:300.

C

Figure 33C. LBR pull down using HA-tagged M50/p35. 293 cells were mock transfected, transfected with HAM50 or co-transfected with HAM50 and wt M53. Total cell lysates were analyzed by SDS-PAGE (12% SDS-gel) and Western blot using specific LBR antibody (T; lanes 1-3). Proteins complexed with HAM50 were precipitated with HA-matrix sepharose beads and eluted proteins were separated by SDS-PAGE (12% SDS-gel). Signals for LBR were visualized by Western blot using specific antibody against LBR (B; lanes 4-8).

The LBR band at 58 kDa was present in all total cell lysates of both sets (Fig.

33C, D, lanes 1-3). Due to a higher resolution of the SDS-PAGE for total cell lysates of the Flag set an additional LBR band was detectable (Fig. 33D, lanes 1-3). Proteins bound to Flag-or HA-matrix were eluted from the respective sepharose beads by SDS-sample buffer, separated by SDS-PAGE and subjected to Western-blot analysis using LBR-specific antibody at a dilution of 1:300.

The signal at 58 kDa, characteristic for the LBR complex major component p58, was detectable after Flag-pull down assay with the lysate of isolated expressed Flag-tagged M53. Due to the higher efficiency compared to the Strep-tag pull down assay p58 was also detectable after HA-pull down with the lysate of isolated expressed HA-tagged M50, indicating direct interaction with both NEC proteins (Fig. 33C, D, lanes 5). The lower LBR complex band was not detectable after Flag-pull down, suggesting two LBR isoforms.

D. Results D

Figure 33D. LBR pull down using Flag-tagged M53/p38. 293 cells were mock transfected, transfected with FlagM53 or co-transfected with FlagM53 and wt M50. Total cell lysates were analyzed by SDS-PAGE (15% SDS-gel) and Western blot using specific LBR antibody (T; lanes 1-3). Proteins complexed with FlagM53 were precipitated with Flag-matrix sepharose beads and eluted proteins were separated by SDS-PAGE (15% SDS-gel). Signals for LBR were visualized by Western blot using specific antibody against LBR (B; lanes 4-8).

In the HA-pull down, the intensity of the LBR signal was increased for co-expressed HAM50 and wt M53 (Fig. 33C, lane 6). The LBR signal intensity was not increased after pull down with co-expressed FlagM53 and wt M50, suggesting a more prominent role of M50/p35 in LBR interaction (Fig. 33D, lane 6).

12. Both M53/p38 and M50/p35 specifically interact with nuclear matrix