Figure 4.

Interaction of the Mutated and Normal DNAL1 Protein with the Dynein Heavy Chain and with Tubulin

(A) Schematic representation of the interaction of DNAL1 with the dynein heavy chain (DNAH) and tubulin.^31,32

(B) Immunoprecipitation of dynein heavy chain. (a) Immunoprecipitation of the dynein heavy chain with the mutated (MUT) and normal (WT) DNAL1-Myc by Myc antibody probed with an anti-dynein heavy chain antibody. (b) The beads were incubated solely with the lysate mixture and axoneme and probed with an anti-dynein heavy chain antibody, thus presenting the background dynein heavy chain signal in the immunoprecipitation. (c) Input quantity of DNAL1-Myc for the reaction. (d) Input quantity of dynein heavy chain extracted from rat tracheas. The percentage in brackets refers to the loading on the gel of the lysate mixture with axoneme relative to the quantity used for the immunoprecipitation. The antibody used for the Immunoblot analysis is denoted at the right side.

(C) Immunopercipitation of α-tubulin. (a) IP of α-tubulin with the mutated (MUT) and normal (WT) DNAL1-Myc by Myc antibody probed with an anti-α-tubulin antibody. (b) The beads were incubated solely with the lysate mixture and axoneme and probed with an anti-α-tubulin antibody, thus presenting the background of α-tubulin signal in the immunoprecipitation. (c) Input quantity of α-tubulin for the reaction. (d) Input quantity of DNAL1. The percentage in brackets refers to the loading on the gel of the lysate mixture with axoneme relative to the quantity used for the immunoprecipitation. The antibody used for the Western analysis is denoted at the right side.

The stability of the mutated DNAL1 after the incubation with the axonemal extracts and with the primary antibody at 4°C was comparable to the normal DNAL1 (not shown).