Figure 2. FRMD8 loss reduces mature ADAM17 levels and impairs ADAM17-dependent shedding activity.

(A) ADAM17 levels were analysed in HEK293T cells transfected with non-targeting siRNA control pool (ctrl) or FRMD8 SMARTpool siRNA after western blotting with anti-ADAM17 and anti-actin staining. In this and subsequent figures, pro- and mature form of ADAM17 are indicated with black and white arrowheads, respectively. Lower panel: Knockdown efficiency of FRMD8 was analysed by TaqMan PCR. (B, C) Lysates from wild-type (WT) and FRMD8 knockout (KO) HEK293T cells, transiently transfected with FRMD8-V5 for 72 hr (where indicated) and immunoblotted for endogenous ADAM17, ADAM10, FRMD8 and actin using western blotting. Nonspecific bands are marked with an asterisk. (D) Cell surface levels of endogenous ADAM10 and ADAM17 were analysed in WT and FRMD8 KO HEK293T cells after stimulation with 200 nM PMA for 5 min. Unpermeabilised cells were stained on ice with ADAM10 and ADAM17 antibodies, or only with the secondary antibody as a control (grey). The immunostaining was analysed by flow cytometry. The graph shown is one representative experiment out of four biological replicates. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test. (E, F) WT and FRMD8 KO HEK293T cells were transiently transfected with alkaline phosphatase (AP)-tagged AREG, HB-EGF or TGFα, and then either incubated with 200 nM PMA, with 200 nM PMA and 1 µM GW (ADAM10/ADAM17 inhibitor), or with DMSO for 30 min. In addition, cells transfected with AP-TGFα were either left unstimulated for 20 hr or incubated with GW for 20 hr. AP activity was measured in supernatants and cell lysates. Each experiment was performed in biological triplicates. The results of three independent shedding experiments are shown. Statistical analysis was performed of using a Mann-Whitney test. ns = p value>0.05; *=p value<0.05; ***=p value<0.001; ****=p value<0.0001.