Supplementary Figure 10: The SH3PXD2A-HTRA1 fusion, but not each of the wild-type gene partners, increase proliferation.

(a) Transient transfection of wild-type HTRA1, SH3PXD2A, SH3PXD2A-HTRA1 fusion and SH3PXD2A-HTRA1 S322A fusion in human Schwann cells. Error bars, s.e.m.; n = 5. (b) Trypan blue direct cell count using automated cell counter 96 h post-transfection. Error bars, s.e.m.; n = 5. (c) Anoikis assay for control and fusion-positive cells with viability measured 12 h post 48 h incubation in non-coated plates to prevent attachment. Error bars, s.e.m.; n = 3. (d) Immunoblot confirming HTRA1, SH3PXD2A single and dual knockdowns in human Schwann cells treated with control siRNA, pooled HTRA1, pooled SH3PXD2A or dual siRNA. (e) Direct cell count of human Schwann cells treated with siRNA from d. (f) Immuno-precipitation (IP) of wild-type and fusion protein. HEK-293 cells were transfected with myc-tagged HTRA1, HA-tagged SH3PXD2A-HTRA1 fusion or HA-tagged SH3PXD2A-HTRA1 S322A (a catalytic-dead protease). Following MYC and HA IPs, proteins were purified and eluted using HA or myc peptides. (g) Protease assay, 100 ng of Immuno-precipitated HTRA1, fusion, fusion dead protein or no protein was incubated with 10 μg of bovine serum albumin (BSA) overnight and run on a 10% gel. Samples were also incubated with protease inhibitors (Roche, Protease Inhibitor Cocktail) to inhibit protease activity. Cropped version of this blot is shown in Figure 5j.