FIGURE 5.

PCSK3/furin cleaves fCTRP12 at the K⁹⁰KSR motif. A and B, relative mRNA levels of different PC family members (PCSK1–7) in epididymal fat tissue (A) and in differentiated 3T3-L1 adipocytes (B). All quantitative real-time PCR values were normalized to 18 S rRNA. The expression of PCSK1 was set to 1, and expression levels of other PC family members were normalized against PCSK1. C, immunoblot analysis and quantification of furin expression in 3T3-L1 preadipocytes, mature 3T3-L1 adipocytes, and adipose tissue (mouse epididymal fat pad). Quantification of furin expression was normalized to β-tubulin. D, immunoblot blot analysis and quantification of furin expression in adipocytes treated with insulin (10 nm) for various time points. E, comparison of furin expression in HEK 293T cells and 3T3-L1 adipocytes. F, HEK 293T cells were co-transfected with wild-type (WT) CTRP12 or its 3M mutant construct and an increasing amount of plasmid DNA encoding PCSK3/furin (lanes 2–9). As a control, HEK 293T cells were also transfected with empty pCDNA3.1 plasmid (lane 1). Shown is the immunoblot analysis of secreted WT or 3M mutant protein found in the supernatant of transfected cells. NS, nonspecific band. G, knockdown of furin in adipocytes resulted in decreased endogenous CTRP12 cleavage. 3T3-L1 adipocytes were infected with lentiviruses encoding an empty vector or shRNA against furin. *, p < 0.05; **, p < 0.01. Error bars, S.E.