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

Figure 2

Enhanced secretion of MMP-28 by co-transfection of COS-1 cells with furin cDNA. The data show that MMP-28 secretion requires coexpression with furin. Furin does not increase newly synthesized MMP-28. Instead, Furin promotes existing MMP-28 secretion from storage pools to secretory pathway. Active state of furin is not prerequisite.

(a) Western blotting of the conditioned medium and cell lysate using anti-Myc antibody: COS-1 cells were cotransfected with MMP-28/Myc chimeric cDNA along with GFP control, or furin cDNAs. The conditioned medium and cell lysate were examined by Western blotting using anti-Myc antibodies. An aliquot of total cell lysate for 𝛼/𝛽 tubulin was used as a loading control

(b) Real-time RT PCR of MMP-28: Total RNA was extracted from COS-1 cells transfected with GFP control, MMP-28, and MMP-28 along with furin cDNA. Expression of MMP-28 mRNA was examined by real-time RT PCR and expression level was normalized by housekeeping genes (HKGs) including HPRT and G6PDH. Experiment was repeated three times with triplicate samples for each category. Each bar represents the mean ± SEM

(c) Western blotting of the conditioned medium using anti-Myc antibody: COS-1 cells cotransfected with MMP-28 cDNA along with wild type and mutant furin cDNAs. The conditioned medium was examined by Western blotting using anti-Myc antibodies

(d) Western blotting of the conditioned medium and cell lysate using anti-Myc antibody: COS-1 cells were transfected with furin along with wild type and mutant MMP-28. The conditioned medium and cell lysate were examined by Western blotting using anti-Myc antibodies. An aliquot of total cell lysate for 𝛼/𝛽 tubulin was used as a loading control