Figure 3. Disp is cleaved by Furin.

(A) NIH3T3 cells expressing DispHA were treated with increasing concentrations of Furin Inhibitor I (10, 25, 50, 75 and 100 μM). The bracket indicates the 250 kDa fraction. Graph shows Disp densitometry analysis normalized to Kinesin for two independent experiments. Normalized signal intensity for each DispHA species in treated conditions is shown relative to vehicle control intensity, which was set to 1. (B) CRISPR/Cas9 generated knockout lines for Furin, PACE4, PCSK5 and PCSK7 were transfected with V5DispHA-expression vector, and formation of the ~30 kDa V5 cleavage fragment was monitored by western blot of cell lysates from Clonal line 1. Furin and PCSK7 protein levels were examined by western blot. PACE4 and PCSK5 mutations were confirmed by deep sequencing as in Figure 3—figure supplement 1. (C) Lysates from cells co-expressing wild type or CS mutant V5DispHA and Furin-Myc proteins were examined for Disp cleavage by western blot for the V5 fragment. Co-expression of Furin-Myc reduced total Disp signal (top panel). Gain equalization of the V5 signal in Furin-Myc expressing cell lysates is shown for comparison. (D) Furin-Myc and V5DispHA were co-expressed in HEK293T cells and Furin-Myc was immunoprecipitated from lysates using anti-Myc. Input (left) and immunoprecipitates (right) are shown. (E) Wild type and CS mutant V5DispHA proteins were expressed in LoVo (lacking Furin) or HCT-15 (control) colon carcinoma cells and lysates were analyzed by western blot. Re-expression of Furin in LoVo cells rescued cleavage (lane 4 compared to 2). Kinesin and Tubulin are the loading controls for western blots.

Figure 3—figure supplement 1.

(A) Genotype of PC family knockout clones. Red arrows indicate the Cas9 cleavage site. sgRNA sequence (5’ to 3’) is shown for each targeted gene along with corresponding deletions (black dashes) and insertions (red letters) identified by deep sequencing of knockout clonal lines. Insertion/deletion sizes are indicated in parenthesis and resulting premature truncation amino acid is shown in red. (B) CRISPR/Cas9 generated knockout lines for Furin, PACE4, PCSK5 and PCSK7 were transfected with V5DispHA-expression vector, and formation of the ~30 kDa V5 cleavage fragment was monitored by western blot of cell lysates from Clonal line 2. Furin and PCSK7 protein levels were examined by western blot. Kinesin is loading control. PACE4 and PCSK5 were assessed by deep sequencing as in A.