Identification of SNX9 as the antigen to antibodies giving a shorter FLS phenotype. (A) ScFv3 and 4 both detect a specific band not detected in either control sample (red arrow) by Western blotting (left = 25 µg, right = 100 µg). The band at ∼60 kD in all four blots (yellow arrow) is nonspecific reactivity of the secondary or tertiary antibodies with extracts. (B) Western blots of control and SNX9 or mock-depleted extracts with IgGFls3, IgGFls4, or rabbit-anti SNX9. The ∼70 kD band is lost on SNX9 depletion. (C) Side maximum intensity projections of 1 µm confocal z-stacks of either mock or SNX9-depleted extracts. Immunodepletion with anti-SNX9 antibodies reduces FLS length and is rescued by 20 nM purified SNX9. (D–F) Immunoblock (IB) by specific scFvs and rescue by 20 nM purified SNX9. (D) Side maximum intensity projections of 1 µm confocal z-stacks of FLS grown using extracts immunoblocked with control or specific scFVs. Reductions in FLS length and number can be rescued by addition of purified SNX9. (E and F) Quantification shows the median, quartiles, and range, n = 9–12 imaging regions per condition from two independent experiments. Statistics are in Table S1. (G) TIRFM shows colocalization of SNX9 and VASP with actin and Z-maximal projection of a 1-µm confocal stack of actin in FLS. Scale bars, 10 µm.