4.5. p38 MAPK Silencing by siRNA Transfection

To silence p38 MAPK expression, p38 MAPK specific siRNA (s3586, Life Technologies, Carlsbad, CA, USA) and INTERFERin (PolyPlus-Transfection, Illkirch, France), as transfection reagent, were used according to the manufacturer’s instructions.

To silence p38 MAPK, 2.1 × 10⁵ cells/6 mL were seeded into Petri dishes (Ø 6 cm). After 24 h (allowing cells to attach), the culture medium was replaced with new culture medium with or without the p38 MAPK specific siRNA and transfection reagent. The siRNA and transfection reagent were diluted in Opti-MEM^® Reduced Serum Medium (Life Technologies) to a final concentration of 150 nM and 0.4%, respectively, prior to transfection. After 72 h of incubation, cells were harvested and seeded into six-well plates at a density of 1 × 10⁶ cells/2.5 mL per well. After 24 h (allowing cells to attach), the culture medium was replaced with a serum-free medium containing: (1) fresh siRNA, transfection reagent (at the same concentration used for the initial inhibition of p38 MAPK expression) and 2% BSA with or without SA; or (2) 2% BSA with or without SA (control media). After 18 h of treatment, cells were harvested and lysates were prepared for western blot analysis as described previously [8]. The efficiency of p38 MAPK expression silencing was tested in each experiment at the protein level using Western blot analysis.