Figure 2.

General Characterization of Serpina3c in Adipocytes

(A–C) Serpina3c expression patterns induced by MDI or CM treatment in 3T3-L1 adipocytes. Cell lysate were prepared at various times, (A) 0∼6 h or (B) 0∼120 h after treatment for RT-PCR analysis of Serpina3c, Serpina3n, and Gapdh. (C) Also, the expression of adipokines, Leptin and AdipoQ, was compared with those of Serpina3c and Serpina3n.

(D) Tissue distribution of Serpina3c by RT-PCR in C57BL/6 male mice.

(E) Amino acid sequence of Serpina3c was analyzed by SignalP-4.1 prediction (www.cbs.dtu.dk/services/SignalP-4.1/) showing the N-terminal signal peptide for secretory proteins.

(F) Overexpression vector was generated by inserting mouse Serpina3c (or Serpina3n) encoding a C-terminal FLAG tag into pcDNA3.0. HEK293T cells were transfected with this construct, and 2 days later, the medium was collected, precipitated by cold acetone, and subjected to western blot analysis to detect the FLAG-tagged protein secreted from cells. pcDNA3.0 was used as a negative control.

(G) Serpina3c expression and secretion patterns induced by MDI in 3T3 L1 adipocytes. RT-PCR was performed to detect expression of Serpina3c mRNA after treatment. Western blot analysis was performed to investigate expression and secretion of Serpina3c protein in precipitated media or cell lysate after treatment.