Fig. 5.

Loss and recovery of shedding activity after ADAM10 depletion by GI. a THP-1 cells were incubated with 10 μM GI or vehicle control and treated with pHrodo E. coli particles to induce CXCL16 expression or left untreated for 24 h. After washing, cells were incubated for another 24 h with pHrodo E. coli particles in the presence or absence of inhibitor and released CXCL16 was quantified in the conditioned media by ELISA. b HEK293 cells transfected with a betacellulin (BTC) alkaline phosphatase (AP) fusion protein were pretreated with 10 μM GI or vehicle control for 30 min and then stimulated with 1 μM ionomycin to increase ADAM10 activity or left unstimulated. After 45 min, the ratio of released to cell expressed BTC was determined by means of an AP activity assay. c BTC-AP transfected HEK293 cells were incubated with 10 μM GI or vehicle control for 10 min or 24 h. The inhibitor was removed by washing and cells were studied for ionomycin-induced BTC release as in b. d BTC-AP transfected HEK293 cells were incubated with 10 μM GI or vehicle control for 24 h. Cells were washed and either further incubated for 24 h to recover from inhibitor treatment or not allowed to recover (CHX treatment), before they were studied for ionomycin-induced BTC shedding as in b. Data in b–d were normalized to the ionomycin treated control. Data are shown as means and SD of three independent experiments