Constructs and transfection
The complementary DNA (cDNA) of the mouse Sharpin gene was cloned and amplified from RAW264.7 RNA extracts. The primer sequences were forward, 5′-CC ATG GCG ATG TCG CCG CCC GCC GGC GGT; reverse, 5′- AAG CTT CTA GGT GGA AGC TGC AGC AAG A. The Sharpin cDNA was cloned into expression vector pFLAG-CMV-2. Murine fibroblasts and macrophages were were used to express recombinant SHARPIN protein. Cells (2×104) were seeded in 96-well treated plates the day before transfection. After overnight incubation, 200 ng pFLAG-SHARPIN plasmids were transfected with 0.5 ul Lipofectamine 2000. 24 hours later, cells were incubated with fresh culture medium. After another 24 hours, cells were lysed to confirm the FLAG-SHARPIN expression by immunoblots with anti-FLAG. Cells with no transfection and transfected with empty vector pFLAG-CMV-2 were used as negative control in all transfection experiments.