BBS3 pathogenic variants possess abrogated nucleotide binding activities.A, Coomassie-stained SDS gel showing induction of GST alone, untagged wild-type ARL6, and the T31M, T31R, and G169A ARL6 variants. B, Coomassie-stained SDS-polyacrylamide gel showing the proportion of total cell lysate (T) that remains in the soluble fraction (S) of samples from A after a 3-h induction. C, aliquot of the soluble fractions from B was spotted onto nitrocellulose and probed with [α-32P]GTP. GST, the negative control, does not bind nucleotide, whereas ARL6 binds. ARL6(T31R) binds significantly more than wild-type ARL6, and the T31M and G169A variants bind very poorly. D, TLC showing the nature of the radiolabeled nucleotide bound to soluble untagged ARL6 or variant forms of ARL6. Wild-type (untagged) ARL6 binds both GDP and GTP, but the untagged mutants (T31M and T31R) primarily bind GDP. E, Coomassie-stained SDS gel showing pellet fractions wild-type and variant forms (T31M, T31R, G169A, L170W) of the ARL6 protein, induced similarly to A in a separate experiment. F, as with E except that following SDS-PAGE the protein was transferred to nitrocellulose membrane, allowed to re-fold, and probed with [α-32P]GTP.