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. 2006 May;26(10):3917–3934. doi: 10.1128/MCB.26.10.3917-3934.2006

FIG. 3.

FIG. 3.

ADAM 10 is involved in S2 processing. (A) RPTPκ subunit dissociation does not contribute to TFP-induced or basal shedding. RPTPκ constructs used here are HA tagged at the C terminus. COS-7 cells were transfected with cDNAs of RPTPκ, RPTPκ-LNTR, or pRK5 vector (M). Left panel, cells were washed, incubated in serum-free medium, and stimulated with 100 μM TFP for the times indicated. Upper panel, cell lysates were blotted and probed with anti-HA antibody. Lower panels, detection of shed RPTPκ fragments. Conditioned media were collected, and proteins were concentrated by TCA precipitation and probed with anti-RPTPκEC antibody. Right panel, time course of RPTPκ basal shedding at a high cell density. Transfected COS-7 cells were incubated in serum-free medium for the times indicated in the text. Conditioned media were processed as described above. (B) Metalloprotease inhibitor BB-94 diminishes S2 cleavage and shedding of RPTPκ in 786-O and Caki-1 cells. Prior to stimulation, cells were washed and serum-free medium was added. Cells were pretreated either with (+) or without (−) the metalloprotease inhibitor BB-94 (5 μM) or dimethyl sulfoxide (DMSO) and were then stimulated with 100 μM TFP for 15 min. Upper panel, RPTPκ was immunoprecipitated with anti-RPTPκJM antibody. Lower panel, conditioned media were collected, and proteins were concentrated by TCA precipitation, blotted, and probed with anti-RPTPκEC antibody. (C) ADAM 10 is involved in TFP-induced S2 cleavage. Caki-1 cells were transfected with siRNA (siR) directed against ADAM 10 (A10), ADAM 15 (A15), and ADAM 17 (A17). After 48 h, cells were treated or not treated (−) with 100 μM TFP for 15 min. Upper panel, RPTPκ was immunoprecipitated with anti-RPTPκJM antibody. Lower panels, specific silencing of ADAM expression was confirmed by immunoblot analyses of whole-cell lysate with antibodies to ADAM 10, ADAM 15, and ADAM 17. While immature and mature isoforms were detected for ADAM 10 and ADAM 17, no mature isoform was observed for ADAM 15. (D) ADAM 10-mediated cleavage at a high cell density. RPTPκ was immunoprecipitated from confluent ADAM 10+/+ and ADAM 10−/− fibroblasts. Abbreviations: α, anti; B, blotting; IP, immunoprecipitation; WCL, whole-cell lysate.