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. 2023 Jan 13;34(2):br2. doi: 10.1091/mbc.E22-11-0524

FIGURE 1:

FIGURE 1:

DRC2 and DRC4 mostly move independently of each other. (A) TIRF, bright field (BF), and merged images showing a mixture of double mutant–single rescued cells (ida6 pf2 DRC2-GFP and ida6 pf2 DRC4-mC) and double mutant–double rescued cells (ida6 pf2 DRC2-GFP DRC4-mC). TIRF, original frame from the TIRF recording with the GFP signal projected on the left side and the mC signal onto the right side of the chip. Bar = 10 μm. (B) Western blot analyzing flagella isolated from wild type (control), ida6 DRC2-GFP, ida6 pf2 DRC2-GFP, and ida6 pf2 DRC2-GFP DRC4-mC. Membranes 1 and 2 are replicate membranes with equal sample loading. Membrane 1 was first stained with anti-DRC2 (1), followed by anti-IC2 (2) as a loading control, and finally anti-DRC4 (3). Membrane 2 was stained first with anti-GFP (1) and then anti-mC (2). The positions of tagged and endogenous DRC2 and DRC4 and of the marker proteins are indicated. DRC4-mC ran as multiple bands, of which the middle one was the most prominent with both anti-mC and anti-DRC4. The mC tag tends to fragment upon sample heating, likely explaining the banding pattern (Gross et al., 2000). (C) Western blot analyzing flagella isolated from wild type (control), pf2-4 DRC4-mC, ida6 pf2 DRC4-mC, and ida6 pf2 DRC2-GFP DRC4-mC. Membranes 1–4 are replicate membranes with equal loading. Membrane 1 was first stained with anti-DRC4 (1), followed by anti-IC2 (2) as a loading control. Membrane 2 was stained with anti-GFP, membrane 3 with anti-DRC2, and membrane 4 with anti-mC. The positions of tagged and endogenous DRC2 and DRC4 and the marker proteins are indicated. (D) Violin plot of the swimming velocity of wild type, ida6 DRC2-GFP, pf2 DRC4-mC, ida6 pf2 DRC2-GFP, ida6 pf2 DRC4-mC, and ida6 pf2 DRC2-GFP DRC4-mC. The number of cells analyzed is indicated; error bars indicate SD. (E) Violin plot depicting the flagella length distribution in live cells from wild type, ida6 DRC2-GFP, pf2 DRC4-mC, ida6 pf2 DRC2-GFP, ida6 pf2 DRC4-mC, and ida6 pf2 DRC2-GFP DRC4-mC (see Materials and Methods for details).