Figure 3. Interactions between CA tubes and cellular Nups.

(A) CA tubes were assembled in vitro and incubated with lysates of HeLa cells without (lanes 1–5) or with (lanes 6–10) Dox dependent expression of MX2. The reaction mixtures were subjected to centrifugation to separate pulled-down (or bound) fractions from supernatant (unbound) proteins. Lanes 1 and 6: cellular lysates; Lanes 2 and 7: supernatants from control experiments without CA tubes; Lanes 3 and 8: supernatants after incubating cellular lysates with CA tubes; Lanes 4 and 9: pulled-down fractions from control experiments in the absence of CA tubes; Lanes 5 and 10: proteins bound to CA tubes. Bands shown in each blot correspond to those indicated in Figure 2. Representative of two independent experiments. (B) Schematic representation of the nuclear pore complex listing individual members of each subcomplex tested in (A).

Figure 3—figure supplement 1. Specificity of CA binding assay.

(A) Coomassie stained SDS-PAGE image for pull-down experiments with in vitro assembled CA tubes and HeLa cell lysates. Lanes 1–10 correspond to those in Figure 3. In addition, molecular weight markers are shown. Representative of two independent experiments. (B) WT and N74D CA tubes were assembled in vitro and incubated with lysates of HeLa cells without (lanes 1–7) or with (lanes 9–15) Dox dependent expression of MX2. The reaction mixtures were subjected to centrifugation to separate pulled-down (or bound) fractions from supernatant (unbound) proteins. Lanes 1 and 9: cellular lysates; Lanes 2 and 10: supernatants from control experiments without CA tubes; Lanes 3 and 11: supernatants after incubating cellular lysates with WT CA tubes; Lanes 4 and 12: supernatants after incubating cellular lysates with N74D CA tubes; Lanes 5 and 13: pulled-down fractions from control experiments in the absence of CA tubes; Lanes 6 and 14: CPSF6 bound to WT CA tubes; Lanes 7 and 15: No CPSF6 bound to N74D CA tubes. Representative of two independent experiments.