PMC

A: Furin and TGF-β1 mRNA expression in hematopoietic/immune cells and tissues. B: Furin and TGF-β1 mRNA expression in mice tissues. Northern blot analysis used total mRNA (5 μg/lane) and a rat TGF-β1 or a rat furin riboprobe. Ethidium bromide staining of 18S is shown as a control for mRNA integrity.

Co-regulation of furin and TGF-β1 mRNAs. NRK-49F cells were incubated for 4, 6, or 8 hours in the presence or absence of 10 μmol/L PMA or 5 ng/ml human recombinant TGF-β1. Total mRNAs (10 μg/lane) were probed with rat riboprobe specific for furin, TGF-β1, PC7, and a GAPDH cDNA.

PC mRNA regulation by TG F-β1. The rat insulinoma cell line (Rin m5F) (A) or rat fibroblastic kidney cell line (NRK-49F) (B) were incubated for 7, 15, and 24 hours in the presence or absence of 5 ng of recombinant human TGF-β1. Total mRNAs (10 μg/lane) were probed with rat riboprobes specific for furin, PC1, PC5A/PC5B, PACE4, or GAPDH cDNA.

A: Inhibition of TGF-β1 processing by α₁-PDX. BSC-40 cells were infected with vaccinia recombinants for TGF-β1 precursor (VV: TGF-β), control vaccinia virus (VV: WT), furin-encoding vaccinia virus (VV: FUR) and α₁-PDX-encoding vaccinia virus (VV: α₁-PDX) at the indicated multiplicity of infection. Eighteen hours after cell infection, cell supernates were collected and electrophoresed on 12% SDS-PAGE gels under reducing conditions. Immunoblots were performed with an anti-human LAP, PAN-specific anti-TGF-β antibodies, furin-specific antisera (revealing an ∼95-kd form) and α₁-antitrypsin-specific antisera (revealing an ∼55-kd α₁-PDX form). B: Measure of TGF-β1 in cell supernates. Eighteen hours after cell infection, cell supernates were collected, heat-activated (80°C, 5 minutes), and used to quantitate bioactive TGF-β1 as described in Material and Methods. A representative experiment out of two performed is shown.

A: Processing of TGF-β1 precursor by PCs in BSC-40 cells. Cells were infected with vaccinia recombinants for both the TGF-β1 precursor (VV: TGF-β) or an unrelated control vaccinia recombinant (VV: POMC) and one of the PCs (PC1/PC3, VV: PC1; PC2, VV: PC2; PC5A, VV: PC5A; furin, VV: FUR, PACE-4, VV: PACE-4; PC5B, VV: PC5B) at a multiplicity of infection of 5. Eighteen hours after infection, supernates were collected, concentrated, and electrophoresed on 12% SDS-PAGE gels under reducing conditions. Immunoblots were performed with an anti-human LAP IgG (revealing an ∼55-kd proTGF-β1 and an ∼40-kd proregion forms) or PAN-specific anti-TGF-β antibodies (revealing an ∼12-kd mature TGF-β1 form). A representative experiment out of three performed is shown. B: Measure of TGF-β1 in cell supernates. Eighteen hours after cell infection, cell supernates were collected, heat-activated (80°C, 5 minutes), and used to measure bioactive TGF-β1. A representative experiment out of three performed is shown.

A: Processing of TGF-β1 precursor by PCs in LoVo cells. LoVo cells were infected with vaccinia recombinants for both the TGF-β1 precursor (VV: TGF-β) or an unrelated control vaccinia recombinant (VV: POMC) and one of the PCs (PC1/PC3, VV: PC1; PC2, VV: PC2; PC5A, VV: PC5A; furin, VV: FUR, PACE-4, VV: PACE-4; PC5B, VV: PC5B; PC7, VV: PC7) at a multiplicity of infection of 5. Eighteen hours after infection, supernates or cell lysates were electrophoresed on 12% SDS-PAGE gels under reducing conditions. Immunoblots were performed with an anti-human LAP IgG (revealing an ∼55-kd proTGF-β1 and a 40-kd proregion forms) or PAN-specific anti-TGF-β antibodies (revealing an ∼12-kd mature TGF-β1 form). A representative experiment out of four performed is shown. B: Measure of TGF-β1 in cell supernatants or cell lysates. Eighteen hours after cell infection, LoVo cell supernates were collected, heat-activated (80°C, 5 minutes), and used to measure bioactive TGF-β1. A representative experiment out of four performed is shown.