Fig. 2. Biochemical confirmation of lamin-titin binding using three independent assays. (A) Titin and lamins interact in Ni²⁺ pull-down assays. ³⁵S-titin 1-551 was incubated with His-tagged wild-type pre-lamin A tail, and pelleted with Ni²⁺-NTA agarose beads; 20% of each supernatant and 50% of each pellet were resolved by SDS-PAGE (12% gels) and exposed to X-ray film (autoradiograph is shown). (B) Results of the microtiter-well binding assays, in which either the purified pre-lamin A tail or BSA were immobilized in microtiter wells and incubated with 1 nM ³⁵S-titin 1-551 (see Materials and Methods). The data are representative of five independent experiments, each done in triplicate. (C) Binding to lamin-AffiGel beads. Purified recombinant pre-lamin A tail (wild type or mutants; `prog' indicates pre-lamin A with the 50-residue deletion that causes HGPS) or lamin B1 tail were coupled covalently to AffiGel beads, incubated with ³⁵S-titin 1-551 and pelleted to recover bound probe. Pellets (50% of each sample) were resolved by SDS-PAGE and autoradiographed (³⁵S-titin; upper panel) or immunoblotted with antibodies against the His-tag on each lamin tail (lower panel). (D) Quantification of data from panel C plotted as % of each probe that bound and normalized against titin binding to wild-type pre-lamin A tail. Bars indicate standard deviations from four independent experiments.