Figure 1.

GFP-tagged and endogenous α-tubulin have similar properties. (A) Bright-field (a) and TIRF (b and c) images of a live cell expressing GFP–α-tubulin. The two focal planes show cilia (b) and cell body microtubules (c). Bar, 1 µm. (B) Western blot analysis of wild type (WT) and the GFP–α-tubulin expressing strain GFP-Tub1. Whole cells (WC), cell bodies (CB), isolated cilia (FLA), axonemes (AX), and MM fractions were loaded and probed with antibodies to GFP and α-tubulin. The bands corresponding to α-tubulin, GFP–α-tubulin, and the uncleaved ble-GFP–α-tubulin are marked. (C) Western blots probed with anti–α-tubulin and anti-GFP showing different dilutions of the cell body (CB) sample in comparison to the undiluted cilia sample (FLA). The amounts of endogenous and tagged tubulin in cilia correspond to ∼20 and ∼10% of the respective cell body tubulin. (D) Western blot showing a dilution series of axonemes (AX) and undiluted MM of strain GFP-Tub1 to determine the distribution of tagged and endogenous tubulin inside cilia. The blot was probed with antibodies to α-tubulin and the matrix protein IFT139. (E) Western blots of wild-type and GFP-Tub1 axonemes probed with antibodies to α-tubulin, acetylated α-tubulin (6-11B-1), polyglutamylated tubulin (GT335), GFP, and, as a loading control, IC2, an outer arm dynein intermediate chain. (F and G) Schematic presentations of the distribution of GFP-tagged tubulin and endogenous tubulin in whole cells (F) and cilia (G).