Figure 3. ARL13B^V358A is undetectable in cilia and cannot be enriched by inhibition of retrograde transport.

(A) Antibodies against ciliary markers acetylated α-tubulin or IFT88 (magenta) and ARL13B (cyan) in Arl13b^V358A/V358A MEFs. Representative images show staining for five indicated ARL13B antibodies: (PT) polyclonal rabbit antibody against full-length human ARL13B from ProteinTech, (503, 504, 505) polyclonal rabbit sera from three distinct rabbits raised against C-terminus of mouse ARL13B (amino acids 208–427) (Caspary et al., 2007), and (NM) monoclonal mouse antibody against C terminus of mouse ARL13B from NeuroMab. Arl13b^+/+ and Arl13b^V358A/+ show ciliary ARL13B staining. Table lists ARL13B-positive cilia and the total number of cilia identified by acetylated α-tubulin or IFT88 antibody in parentheses. Cilia appear shorter in Arl13b^V358A/V358A cells (see Figure 6). (B) IFT88 and (C) Gli3 (green) is enriched in the tips of cilia in Arl13b^+/+ (+/+), Arl13b^V358A/+ (A/+) and Arl13b^V358A/V358A (A/A) cells following ciliobrevin-D treatment. Violin plots depict relative fluorescence of IFT88 and Gli3 at cilia tip to cell body with number of cilia measured (n) listed beneath each plot. (D and E) Table lists ARL13B (cyan) positive cilia (rabbit anti-ARL13B; ProteinTech or mouse anti-ARL13B; NeuroMab) and the total number of cilia (acetylated α-tubulin: magenta) examined in control (DMSO) and ciliobrevin-D treated (30 μM CB) cell lines. Representative images show staining for cilia and ARL13B. Staining of IFT88 and Gli3 analyzed by two-way ANOVA and Sidak’s multiple comparisons. (***p<0.001, ****p<0.0001).

Figure 3—figure supplement 1. Endogenous ARL13B is undetectable in the cell body of cilia mutant Ift172^wim/wim cells.

Immunofluorescent detection of ARL13B (cyan; NeuroMab) and cilia marker acetylated α-tubulin (magenta) in wildtype, Arl13b^hnn/hnn, Ift172^wim/wim, and Ift172^wim/wim Arl13b^hnn/hnn cell lines. (A) In wildtype cells, ARL13B is detectable in the cilium under non-saturating conditions, these parameters are kept constant across all samples. (B) In Ift172^wim/wim non-ciliated cells, ARL13B protein is confined to the cell body, but is nearly undetectable by standard immunofluorescence. (C–D) In Arl13b^hnn/hnn and Ift172^wim/wim Arl13b^hnn/hnn cells, null for Arl13b, any signal is due primary antibody background. At an exposure rate 4x above normal the ARL13B signal saturates the detector in wildtype cells. In Ift172^wim/wim cells, overexposure reveals no ARL13B positive stain that is above the background detected in Arl13b^hnn/hnn and Ift172^wim/wim Arl13b^hnn/hnn cells. Images taken at 40x. Scale bar is 5 μm. Experiments repeated in duplicate and examined 4–6 fields of view per condition.