Furin Functions as a Nonproteolytic Chaperone for Matrix Metalloproteinase-28: MMP-28 Propeptide Sequence Requirement

Figure 4

Complex formation between MMP-28 and furin as examined by coimmunoprecipitation and coimmunostaining.

(a) Coimmunoprecipitation assay using anti-Myc antibody: COS-1 cells transfected with MMP-28/Myc along with wild-type and mutant furin cDNAs. The cell lysates were precipitated with Myc monoclonal antibody followed by immunoblotting with furin polyclonal antibodies. An aliquot of total cell lysate before immunoprecipitation for 𝛼/𝛽 tubulin was used as a loading control

(b) Coimmunoprecipitation assay using furin antibody: COS-1 cells transfected with MMP-28 or MT1-MMP along with wild type and mutant furin cDNAs. The cell lysates were precipitated with furin polyclonal antibodies followed by immunoblotting with anti-Myc monoclonal antibody. An aliquot of total cell lysate before immunoprecipitation for 𝛼/𝛽 tubulin was used as a loading control

(c) Coimmunostaining of furin and MMP-28: COS-1 cells cotransfected with MMP-28/Myc and furin cDNAs were fixed and permeablized followed by sequentially incubating with primary antibodies (anti-Myc and antifurin antibodies) and secondary antibodies (anti-mouse IgG-Alexa-568 and anti-rabbit IgG-FITC). The cells were examined under con-focal microscopy