(A–C) Proteins pre-mixed with bacterial extract were subjected to affinity pulldowns with FG-repeats as baits. SYPRO Ruby stained SDS-PAGE gels show (left to right) input mixes, their components (bacterial extract and test proteins, respectively) and proteins eluted with 1M salt from mock or various FG-repeat coated beads. (A) Binding reactions with the NTR Kap95 (positive control) and inert proteins 3xGFP and MBP-GFP-Nup53ΔC (negative controls). Note that only Kap95 can be efficiently purified from the bacterial extract mixtures. (B and C) Similar to (A) FG-repeat binding assays with various portions of (B) Nic96-subcomplex and (C) Nup84-subcomplex.
(D) Results of FG-repeat binding experiments. Binding was considered weak unless the eluted test protein band was comparable to or stronger than the input (test protein).