FIGURE 4.

Cathepsin L cleaves the CTR1 ectodomain in vitro and primes further ectodomain processing. A, diagram of the CTR1 ectodomain purified for in vitro cathepsin cleavage studies and SDS-PAGE of purified ectodomain (Ctr1 ecto). Lane M, molecular mass marker. B, CTR1 ectodomain was incubated with recombinant cathepsin L at the indicated enzyme concentration, and products were resolved by SDS-PAGE and Ponceau S staining. The products generated by cathepsin L cleavage are indicated as 1 or 2. C, site of cathepsin L cleavage (red arrowhead) determined by mass spectrometry and the previously identified amino termini of tCtr1 (blue arrowhead). Asterisks indicate residues within the cathepsin L cleavage site found in several mammalian Ctr1 proteins. D, Ctr1^−/−Ctr2^−/− cells containing a Dox-inducible Ctr2 gene were transfected with either an empty vector (V), a vector containing wild type Ctr1 (WT), or Ctr1 lacking the first 8 residues (Ctr1Δ1–8) before being treated with 100 ng/ml doxycycline for 48 h. Cells were then harvested and processed for immunoblotting with the indicated antibodies.