Fig 1. Immunoblot analyses reveal DYX1C1 is shorter in pf23 and the wild-type DYX1C1 gene rescues the pf23 mutant.
(A) Immunoblotting of whole cell samples from wild-type, pf23, pf23gR (T1, T5, T9, T14) using the anti-DYX1C1 antibody. Wild-type cells express an ~95 kDa DYX1C1 (blue arrowhead), while pf23 expresses an ~92 kDa DYX1C1 (pink arrowhead), because of the loss of 27 amino acids that correspond to the wild-type 5th exon. Rescued strains (T1, T5 and T14) expressed both wild-type and mutant-type DYX1C1. Note that pf23gR-T9 expresses a short and reduced, but partially functional ~67 kDa DYX1C1 (green arrowhead, see text and Fig 3). (B) Immunoblotting of whole cell samples from wild-type, pf23, pf23cR (NT and 3×HA) using the anti-DYX1C1 and anti-HA antibodies. pf23cR-NT strains expressed both wild-type and mutant DYX1C1. pf23cR-3×HA strain expressed mutant and wild-type DYX1C1 tagged with 3×HA epitopes (orange arrowhead). This DYX1C1-3×HA in pf23cR-3×HA was also detected by the anti-HA antibody (orange arrowhead). The membrane, blotted with the DYX1C1 antibody (middle), was re-blotted with the anti-HA antibody (bottom). (C) Restoration of swimming in rescued pf23 strains. Swimming velocity was measured for wild-type and rescued strains of pf23. 20–25 cells were selected for measurement of swimming speed. The original pf23 is completely non-motile, and for the pf23 strain “NM” indicates non-motile/not measured. Note that pf23gR-T9 shows slower swimming compared to wild-type, possibly because of the reduced expression of the truncated DYX1C1 fragment and the resultant partial failure of inner dynein arm assembly (discussed in the text).