Figure 3.

ADAM-12 and MT1-MMP blocking antibodies or siRNA silencing prevent shedding of the LRP-1 ectodomain. A, B) Blocking antibodies: Western blotting of LRP-1 α-chain and GAPDH in lysates of fibroblastoid HT1080 cells treated for 24 h with 10 μg/ml anti-ADAM-12 antibodies (A) or nonimmune goat IgG control (CTRL); or 5 μg/ml anti-MT1-MMP antibody (B) or nonimmune mouse IgG control. C, D) Efficiency and specificity of siRNAs targeting ADAM-12 (C) or MT1-MMP (D). Fibroblastoid HT1080 cells were treated as described under Materials and Methods with 100 nM-specific siRNA or negative controls. Western blotting of ADAM-12, MT1-MMP, LRP-1 α-chain, and GAPDH was performed in cell lysates, and blotting of LRP-1 α-chain in corresponding amounts of 24-h conditioned medium. Data are from a representative experiment for the most effective siRNA against each target. All experiments were performed at least twice in triplicate.