Journal: The Journal of Cell Biology
Article Title: The MIS12 complex is a protein interaction hub for outer kinetochore assembly
Figure Lengend Snippet: Role of the NSL1 C-terminal tail. (A) The DSN1–NSL1 subcomplex is sufficient to bind KNL1 and NDC80C. GST-NSL1–DSN1 complex was purified by glutathione Sepharose affinity purification and used as an affinity bait for pull-down assays. Copurification of DSN1 with GST-tagged NSL1 was assessed by immunoblotting. GST-NSL1–DSN1 specifically binds KNL1 2106–2316 (lane 4) and NDC80C (as judged by immunoblotting on the NDC80 subunit; lane 5), and the binding is not mutually exclusive (lane 6). The levels of unspecific binding to GST are shown in lanes 1 and 2. (B) HeLa cells were transiently transfected with plasmids containing GFP alone, GFP-tagged NSL1, and GFP-tagged NSL1 EE . After 48 h, cells were washed once and treated with 5 µM S-trityl- l -cysteine for 16 h. Mitotic cells were harvested by vigorous shake-off. Immunoprecipitates (IPs) with an anti-GFP antibody were examined by immunoblotting (IB) with the indicated antibodies. Only GFP-NSL1 was able to bind endogenous NDC80. The band indicated by the asterisk may represent a degradation product of the GFP fusion proteins. wt, wild type. (C) HeLa cells were transiently transfected with plasmids containing GFP alone, GFP-tagged NSL1, GFP-tagged NSL1 EE , and GFP-tagged NSL1 258 . After 48 h, cells were washed once, treated with 3.3 µM nocodazole for 14 h, and analyzed by immunofluorescence. Bar, 5 µm. (D) The means of KNL1 or NDC80 kinetochore (KT) intensities normalized to CREST signal in cells transiently expressing GFP alone, GFP-tagged NSL1, GFP-tagged NSL1 EE , and GFP-tagged NSL1 258 from the experiment in C were calculated and plotted. Error bars represent the SEM. For each condition, at least four different cells were used in the quantification.
Article Snippet: Fixed concentrations (10 nM) of fluorescein-labeled NSL1 peptides (synthesized by Mimotopes) were mixed with increasing concentrations of the indicated SPC24–SPC25 or HP1 constructs in PBS buffer, and reaction mixtures were allowed to equilibrate overnight at 4°C.
Techniques: Purification, Affinity Purification, Copurification, Binding Assay, Transfection, Immunofluorescence, Expressing