RNA is required for SFPQ-RNA binding to KIF5A/KLC1. (A) Schematic of the indicated constructs for KLC1. (B) HEK 293T cells transfected with HA-SFPQ with empty vector, full-length WT, or tail-truncated Myc-KLC1. HA was immunoprecipitated and blotted for Myc and HA. (C) Quantification of pull down in B relative to input. **P = 0.0033 by unpaired two-tailed t test; n = 4; data represent mean ± SEM. (D) Schematic of the indicated constructs for KIF5A. KLC binding region is indicated. (E) HEK 293T cells transfected with KLC1-Myc and with either WT FLAG-tagged KIF5A or the ΔTail mutant. Myc was immunoprecipitated and blotted for FLAG and Myc. (F) HEK 293T cells transfected with HA-SFPQ with full-length WT, tail-truncated, or tail only FLAG-tagged KIF5A. HA was immunoprecipitated and blotted for FLAG and HA. (G) Quantification of pull down in F relative to input; **P = 0.0014 by independent Welch’s t test; n = 3; data represent mean ± SEM. (H) HEK 293T cells transfected with HA-SFPQ, KLC1-Myc, and FLAG-KIF5A, and lysates were treated with or without RNase. HA was immunoprecipitated and blotted for HA, Myc, and FLAG. (I) Quantification of pull down of KLC1-Myc or FLAG-KIF5A (bottom) in H relative to input; ****P < 0.0001 by unpaired two-tailed t test; n = 3; data represent mean ± SEM. For B, E, F, and H, input represents 0.5% of 0.5 mg of protein lysate used for IP. Total eluate from the bead was run on the gel. Heptad, Heptad repeat; TPR, tetratricopeptide repeat.