Figure 4. ADAM12 inhibition using ADAM12 prodomain (PA12) reduces HB-EGF shedding in endometriosis cell culture.

(a) Coomassie-stained SDS-PAGE gel showing PA12 isolation from E. coli inclusion bodies (i); its purification from Ni-NTA affinity chromatography (ii); and its subsequent refolding, dialysis, and concentration (iii). (b) PA12 inhibits recombinant ADAM12 but not recombinant ADAM-10 or -17, measured by inferring inhibitory constants (K_i) from dose-response curves in a fluorogenic FRET-peptide based assay. (n = 2 ± SD). (c,d) 2 μM PA12 treatment for 3 h increases relative levels of full-length HB-EGF on the cell surface. 12Z-HE cells stably expressing HB-EGF with Myc-tagged ectodomain and a GFP-tagged cytoplasmic tail were stained, fixed, and analyzed by flow-cytometry. (d) Corresponding to c, 3 h PA12 treatment reduces supernatant accumulation of HB-EGF, measured by ELISA. (e,f) Genetic ADAM12 knockdown increases relative levels of full-length HB-EGF on the cell surface (e), while decreasing its accumulation in the supernatant (f). 12Z-HE cells were treated with siRNA for 72 h, supernatant was exchanged, and 3 h later cells were analyzed by flow cytometry (e) and supernatant HB-EGF was measured by ELISA. (c–f) (n ≥ 3 ± SEM); *p < 0.05; two-tailed student’s t-test.