HeLa cells were transiently co-transfected for 48 hours with 8 μg of plasmid constructs encoding Flag-tagged full-length Bcl-GL or a series of Flag-tagged partial Bcl-GL proteins (FL, N-1, N-2, N-3, N-4, C-1, C-2 and C-3) as well as the same amount of plasmid encoding HA-tagged WT-MELK, using the FuGENE6 transfection reagent (Roche).
Cells were lysed with lysis buffer as described above.
The lysates were pre-cleaned with normal mouse IgG (1.2 μg) and rec-Protein G Sepharose 4B (Zymed, San Francisco, CA, USA) at 4°C for 30 minutes.
Subsequently, the lysate was incubated with anti-Flag agarose M2 gel (Sigma-Aldrich) at 4°C for 12 hours.
After washing three times with lysis buffer, proteins on beads were eluted with SDS sample buffer.
To validate an interaction between WT-MELK and Bcl-GL, we constructed plasmids designed to express HA-tagged WT-MELK (HA-WT-MELK) and Flag-tagged Bcl-GL (Flag-Bcl-GL).
These plasmids were co-transfected into HeLa cells and the proteins immunoprecipitated with anti-Flag antibody.
Immunoblotting of the precipitates using anti-HA antibodies indicated that Flag-Bcl-GL was co-precipitated with HA-WT-MELK (Figure 3c).
To further determine which segment of Bcl-GL can interact with WT-MELK, we performed co-immunoprecipitation analyses using HA-WT-MELK and partial Bcl-GL proteins tagged with Flag (Figure 3e).
After co-transfection of plasmid clones into HeLa cells, we performed immunoprecipitation with an anti-Flag antibody, and then immunoblotting with an anti-HA antibody.
(c) Interaction of MELK with Bcl-GL.
Extracts from HeLa-cells transfected with HA (hemagglutinin)-tagged WT-MELK (HA-WT-MELK) or Flag-tagged Bcl-GL (Flag-Bcl-GL), or a combination of these, were harvested 36 hours after transfection.
The cell lysates were immunoprecipitated with anti-Flag M2 antibody.
Precipitated proteins were separated by SDS-PAGE and western blotting analysis was performed with an anti-HA antibody.
(f) Determination of the WT-MELK binding regions of Bcl-GL by immunoprecipitation.
The HA-tagged WT-MELK and various peptide sequences of Flag-tagged Bcl-GL (Figure 3e) were pulled down by immunoprecipitation with Flag-M2 antibody and then immunoblotted with rabbit anti-Flag antibody.
The expression of HA-tagged WT-MELK in total cell lysates was confirmed by western blotting analysis.
As a control, immunoprecipitation was performed from cells co-transfected with pCAGGSn3FC (Mock) and HA-tagged WT-MELK (HA-WT-MELK) through all steps.PP1 has 3 isoforms encoded by different genes that are 97% conserved across their catalytic domains and distinct roles for each isoform have yet to be determined.
When we coimmunoprecipitated Flag-epitope tagged PP1α, β or γ with BRCA1, we observed that all 3 isoforms interacted with BRCA1.
Additionally, we have identified the functional PP1 interacting domain within BRCA1.
This domain is found in other PP1 regulatory proteins, suggesting that BRCA1 may regulate the activity of PP1 and could act as a scaffold protein to promote the dephosphorylation of BRCA1 associated proteins by PP1.Co-immunoprecipitation assays were used to confirm the protein–protein interactions.
Subsequently, localization of ACBP4 and its interacting protein, AtEBP, was confirmed using transient expression of GFP- and DsRed-tagged fusion proteins in Nicotiana tabacum.