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. 2009 Jul 1;20(13):3044–3054. doi: 10.1091/mbc.E09-04-0276

Figure 3.

Figure 3.

IC97 interacts with both α- and β-tubulin. (A) Western blots of wild-type axonemes that were treated with EDC were probed with the IC97 antibody. A prominent cross-linked product of ∼140-kDa is formed (arrow; uncross-linked IC97 is marked by the arrowhead). (B) The IC97 antibody was used to immunoprecipitate the cross-linked product (arrow; uncross-linked IC97 is marked by the arrowhead). The image is a representative silver-stained gel of the immune complexes showing that the IC97 antibody pulls down both uncross-linked IC97 (arrowhead) and the EDC-generated cross-linked product (arrow). The band corresponding to the cross-linked product was excised from an identical SYPRO Ruby-stained gel and components identified by tandem mass spectrometry. The cross-linked product represents a mixture of IC97 cross-linked to a-tubulin and IC97 cross-linked to β-tubulin. (C) Western blots of the IC97-immune complexes derived from EDC-treated axonemes containing HA-tagged α1-tubulin (Kozminski et al., 1993) were probed with the IC97 and HA antibodies. The IC97 antibody detects both uncross-linked (arrowhead) and cross-linked IC97 (arrow). The HA antibody detects only the cross-linked product indicating that it contains IC97 and HA-tagged α1-tubulin. (D) FPLC-purified I1 dynein was bound to Taxol-stabilized microtubules (assembled from purified tubulin) and then EDC cross-linked. The 140-kDa IC97-cross-linked product is formed indicating that IC97 cross-links to tubulin in vitro. (E) Blot overlays using biotinylated tubulin (left) reveal a tubulin-interacting band at the expected size for IC97 in FPLC-purified I1 dynein fractions (asterisk). Also evident is tubulin binding to IC140/IC138 (diamond) of I1 dynein and IC1 (circle) of the outer dynein arm (King et al., 1991). The IC2 component of the outer dynein arm is observed in the Coomassie-stained gel (right), but it is not detected in the blot overlay. The left lanes (αβ + e) are purified outer dynein arm fractions; the right lanes (f) are purified I1-dynein fractions.