i.p : FLAG- Tspan blot: HA-ADAM10

1. Tspan14 Tspan15 Tspan33 FLAG-Tspan: Wild-type or palmitoylation mutant: - - WT PM WT PM WT PM - HA-ADAM10: + + + + + + + i.p: FLAG-Tspan blot: HA-ADAM10 A 80 58 n.s. n.s. n.s. 200 160 HA-ADAM10 quantitation from FLAG i.p. Relative ADAM10 level 100 50 0 whole cell lysate blot: HA-ADAM10 B 80 58 ** 100 80 HA-ADAM10 quantitation from whole cell lysate 60 % mature ADAM10 40 20 0 46 C i.p: FLAG-Tspan blot: FLAG-Tspan 30 25 Supplementary Figure II.Palmitoylation of Tspan14, Tspan15 and Tspan33 is not essential for ADAM10 interaction or promotion of maturation. The experiment was carried out using transfected HEK293T cells that were surface cross-linked with DTSSP and lysed in 1% Triton X-100 containing 0.1% SDS. Immunoprecipitations and western blotting were performed as described in Figure 2A-C, except that wild-type (WT) and palmitoylation mutant (PM) forms of human tetraspanins were compared (n=3; **p<0.01 compared to ADAM10 only; n.s. indicates data not significant between pairs; error bars represent standard error of the mean).