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. 2009 Apr 24;284(17):11738–11747. doi: 10.1074/jbc.M805894200

FIGURE 2.

FIGURE 2.

ADAM10 ectodomain shedding is mediated by ADAMs 9 and 15. A, Western blots of total MEF cell extracts (CE) (50 μg/lane) and culture supernatants (SN) (30 μg/lane). Metalloprotease inhibitors TAPI-1 (25 μm), TAPI-2 (25 μm), and GM6001 (50 μm) significantly reduce ADAM10 processing. B, Western blots of total MEF cell extracts and supernatants. Levels of ADAM10 expression are similar in WT, ADAM9-/-, or ADAM9/15-/- cells (panel 1). No ADAM9 and ADAM15 protein expression was detected in the respective single and double knockouts (second and third panels). Levels of both ADAM10 CTFs and sADAM10 are strongly reduced in ADAM9-/- MEFs (fourth and seventh panels, lanes 4-6). Residual ADAM10 CTF accumulation and sADAM10 are strongly reduced in combined ADAM9/15-/- MEFs, but not completely abolished as demonstrated by the longer exposure (overnight, O.N.) (fourth and seventh panels, lanes 7-9). Note that the levels of ADAM10 CTFs vary independently of the constant PS1 expression levels (compare fourth and fifth panels). The ADAM10 CTF appears sometimes as a doublet band, potentially related to post-translational modifications or alternative cleavage sites. C, Western blot of total cell extracts and cell supernatant. Untransfected COS cells show the presence of low amounts of endogenously expressed ADAM10 (first panel, lanes 1 and 2) and ADAM9 (second panel, lanes 1 and 2). Overexpression of ADAM9 alone or ADAM9EA does not affect ADAM10 expression (first panel, lanes 3 and 4, respectively). In ADAM10-transfected cells (1 μg), ADAM10 full-length protein, ADAM10 CTFs, and sADAM10 are easily demonstrated (third and sixth panels, lanes 5-7). Cotransfection of ADAM10 with increasing amounts of ADAM9 (second panel, 0.1 μg in lanes 14-15 and 1 μg in lanes 8-10) causes a parallel increase in ADAM10 CTFs and sADAM10 (panels 3 and 6, lanes 14-15 and lanes 8-10, respectively). This increase was not observed when co-transfecting ADAM10 with different concentrations of the catalytically inactive ADAM9EA mutant (third and sixth panels, lanes 16-17 and lanes 11-13). Expression levels of PS1 (fourth panel) do not show major variations. The β-actin Western blot and Coomassie stain reflect equal loading of proteins in both cell extract and supernatant samples (fifth and seventh panels, respectively). D, membrane extracts prepared from 5-day-old (P5), 4-week-old (4wk), and 1-year-old (1y) mice brains show a decrease in ADAM10-CTF formation in ADAM9 and ADAM9/15 knockouts, compared with age-matched WT mice.