Figure 3.
Cdt1 and the Ska1 complex physically interact with each other. (A) Diagrammatic representation of the Cdt1 (Cdt192-546) construct purified from bacteria that were used for in vitro interaction studies with the Ska1 complex. The winged-helix (WH) domains relevant for the purpose of the current study has been depicted. (B) Thymidine synchronized and nocodazole arrested mitotic HeLa cell extracts were incubated either with purified GST or GST-tagged Cdt192-546 proteins (10 μg each), followed by pull down with glutathione agarose beads. UB indicates the unbound or flow through fraction and B indicates the proteins retrieved after elution with reduced glutathione. The blots were probed with anti-GST antibody and SKA1 and SKA3 antibodies. (C) Thymidine synchronized and nocodazole arrested mitotic HeLa cell extracts were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies; IgG was taken as a negative control. (D) Thymidine synchronized and nocodazole arrested mitotic HeLa cells that stably expressing GFP-SKA1 (induced by adding 2.5 µg/ml of doxycycline) were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies; IgG was taken as a negative control. 1% of the lysate was loaded as input. (E) Blot overlay assay to study Cdt1-Ska1 complex interaction. 0.5 µg each of the indicated proteins were loaded as baits (top labels) on 18% SDS-PAG, transferred to nitrocellulose membrane and blocked with 5% SM-TBST. 1 µg of the indicated proteins were overlaid as prey proteins (arrows on the right-side labels) on the membrane for 12 h at 4°C. The blot was washed and probed with the indicated antibodies (bottom labels) followed by chemiluminescence. Arrows depict the proteins of interest and the required molecular mass standards are shown in kD (left labels). (F) Biolayer interferometry (BLItz) sensograms obtained using 0.2 μg/ml of GFP-tagged Cdt192-546-loaded amine-reactive biosensors and increasing concentrations (0.3–10 µM) of the Ska1 complex used as analyte to generate a series of sensograms showing the binding and dissociation phases after the baseline. Binding curves were fit globally to a 1:1 binding model to yield equilibrium dissociation constant (KD) noted above. (G) Linear diagram of the full-length and truncated Cdt1 and SKA1 proteins used to map the Cdt1-SKA1 interaction domains. (H) Table for KD values estimated from the BLI sensograms obtained for the pairwise binding of the indicated purified protein constructs. Source data are available for this figure: SourceData F3.

Cdt1 and the Ska1 complex physically interact with each other. (A) Diagrammatic representation of the Cdt1 (Cdt192-546) construct purified from bacteria that were used for in vitro interaction studies with the Ska1 complex. The winged-helix (WH) domains relevant for the purpose of the current study has been depicted. (B) Thymidine synchronized and nocodazole arrested mitotic HeLa cell extracts were incubated either with purified GST or GST-tagged Cdt192-546 proteins (10 μg each), followed by pull down with glutathione agarose beads. UB indicates the unbound or flow through fraction and B indicates the proteins retrieved after elution with reduced glutathione. The blots were probed with anti-GST antibody and SKA1 and SKA3 antibodies. (C) Thymidine synchronized and nocodazole arrested mitotic HeLa cell extracts were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies; IgG was taken as a negative control. (D) Thymidine synchronized and nocodazole arrested mitotic HeLa cells that stably expressing GFP-SKA1 (induced by adding 2.5 µg/ml of doxycycline) were immunoprecipitated (IP) and immunoblotted (IB) with the indicated antibodies; IgG was taken as a negative control. 1% of the lysate was loaded as input. (E) Blot overlay assay to study Cdt1-Ska1 complex interaction. 0.5 µg each of the indicated proteins were loaded as baits (top labels) on 18% SDS-PAG, transferred to nitrocellulose membrane and blocked with 5% SM-TBST. 1 µg of the indicated proteins were overlaid as prey proteins (arrows on the right-side labels) on the membrane for 12 h at 4°C. The blot was washed and probed with the indicated antibodies (bottom labels) followed by chemiluminescence. Arrows depict the proteins of interest and the required molecular mass standards are shown in kD (left labels). (F) Biolayer interferometry (BLItz) sensograms obtained using 0.2 μg/ml of GFP-tagged Cdt192-546-loaded amine-reactive biosensors and increasing concentrations (0.3–10 µM) of the Ska1 complex used as analyte to generate a series of sensograms showing the binding and dissociation phases after the baseline. Binding curves were fit globally to a 1:1 binding model to yield equilibrium dissociation constant (KD) noted above. (G) Linear diagram of the full-length and truncated Cdt1 and SKA1 proteins used to map the Cdt1-SKA1 interaction domains. (H) Table for KD values estimated from the BLI sensograms obtained for the pairwise binding of the indicated purified protein constructs. Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal