Library of sirnas

HEK293 cells (ATCC) were transfected with a library of siRNAs (Ambion) directed against mRNA of 150 known or predicted human serine proteases (Supplementary Data 1). In the first step, each individual serine protease was targeted by two different siRNAs. Transfection mixes (100 μl) were prepared in opti-MEM medium (Invitrogen, [31985062]) by adding 5 μM siRNAs and 2 μl oligofectamine reagent (Invitrogen, [12252011]). The transfection mixes were then added to the HEK293 cells in the presence of 400 μl of serum free DMEM medium (Final volume=500 μl). After 4 hrs incubation, additional 500 μl of DMEM medium containing 20% FCS, 2% PenStrep (Penicillin Streptomycin) and 2% glutamine were added into the 500 μl serum free culture to constitute the complete DMEM medium. In a second step, the cells were transfected with FLAG-HDAC4 (100 ng) and MYC-PKA (200 ng) and as a control, with HDAC4 (100 ng) and an empty pcDNA 3.0 (200 ng) 36 hrs post siRNA transfection. Finally, cells were harvested and lysed in lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, Protease inhibitor cocktail tablets (Roche), 1 mM PMSF and 1% Triton X-100) for Western blotting analysis 24 hrs post expression plasmid transfection.