Figure 7. FRMD8 loss leads to the destabilisation of ADAM17 and iRhom2.

(A) Unpermeabilised WT (black) and FRMD8 KO HEK293T (cyan) cells stably expressing human iRhom2-3xHA were immunostained on ice for HA. Wild-type HEK293T cells immunostained for HA served as a negative control (grey). (B) Cells were permeabilised and stained at room temperature with an anti-HA antibody. Immunostaining with the Alexa Fluor 488-coupled secondary antibody served as a control (grey). The flow cytometry graphs shown are one representative experiment out of three experiments. The geometric mean fluorescence was calculated for each experiment using FlowJo software. Statistical analysis was performed using an unpaired t-test; ns = p value>0.05; *=p value<0.05. (C) Lysates of HEK293T cells stably expressing human iRhom2-3xHA and transiently transfected with FRMD8-V5 (where indicated) were analysed by western blot for iRhom2 levels using anti-HA, anti-V5 and anti-actin immunostaining. Nonspecific bands are marked with an asterisk. (D) Lysates of WT and FRMD8 KO HEK293T cells stably expressing human iRhom2-3xHA (where indicated) were immunoblotted for HA, FRMD8 and actin. An asterisk marks nonspecific bands. (E) FRMD8 mRNA levels relative to actin mRNA levels were determined by TaqMan PCR in cells used in (D).

Figure 7—figure supplement 1. FRMD8 stabilises iRhom levels by preventing its lysosomal degradation.

(A) iRhom1/2 double knockout HEK293T cells stably expressing iRhom2^WT-3xHA, iRhom2^Δ300-3xHA or FRMD8-iRhom2^Δ300-3xHA were treated with 100 µg/ml cycloheximide (CHX) for the indicated time (0–8 hr) to block protein synthesis. Cell lysates were immunoblotted for HA and actin. (B) Cell lysates of wild-type (WT) and FRMD8 knockout (KO) HEK293T cells treated with 10 µM MG-132 (MG), 200 nM bafilomycin A1 (Baf) or 50 mM ammonium chloride (NH₄Cl) for 16 hr were immunoblotted for ADAM17, FRMD8, and actin. An asterisk marks a nonspecific band. (C) N-glycosylation of iRhom2 was analysed using EndoH and PNGase to distinguish ER/cis-Golgi (EndoH sensitive) and late Golgi localisation (EndoH resistant). Lysates of WT and FRMD8 KO HEK293T cells transiently transfected with mouse iRhom2-3xHA were deglycosylated with EndoH or PNGase and then immunoblotted for mouse iRhom2, human FRMD8 and actin. An asterisk marks a nonspecific band. (D) Lysates of HEK293T cells stably expressing human iRhom1-3xHA and transfected with FRMD8-V5 (where indicated) were immunoblotted for HA, V5, and actin. (E) Levels of ADAM17 were analysed in HEK293T-iRhom2-3xHA and HEK293T WT cells transfected with siRNAs targeting iRhom2 where indicated. Cell lysates were immunoblotted using an anti-ADAM17 or anti-actin antibody. An asterisk marks a nonspecific band.