p38 MAPK, ROCK2, and JNK Phosphorylation Assay

RAW Blue cells (Invivogen) were grown under standard conditions (37°C, 5% CO₂) and maintained under passage number 15. Cells were equilibrated with 1, 5, or 6 (10⁻⁶M) or Kineret (1.0 mg/mL) for 30 min, after which time they were exposed to IL-1β (100 ng/mL) for 15 min. Cells were harvested and lysed on ice for 30 min using a radioimmunoprecipitation assay buffer (Cell Signaling) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and cOmplete™ EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany, prepared according to the manufacturer's instructions). Protein concentrations were determined using a Bradford protein assay (Bio-Rad) on 96-well plates with a microplate reader (EnVision Multilabel reader) measuring OD at 595 mm. Bovine serum albumin serial dilutions were used to generate a standard curve. Lysates were then mixed with 4X reducing sample buffer (Bio-Rad).

Lysates were loaded (30 μg protein per well) in a 5% acrylamide stacking gel, and samples were electrophoresed in a 12% acrylamide resolving gel for 1.5 h at 120 V, followed by a 1 h transfer onto polyvinylidene difluoride (PDVF) membranes at 100 V. Membranes were blocked and incubated with 1:1,000 dilution of primary antibody and 1:20,000 dilution of secondary antibodies according to the manufacturer's instructions. Antibodies used were for phospho-p38 MAPK (Cell Signaling, #9211), p38 MAPK (Cell Signaling, #9212), SAPK/JNK (Cell Signaling, #9252), phospho-SAPK/JNK (Cell Signaling, #9251), ROCK2 (Thermo Fisher Scientific PA5-21131), phospho-ROCK2 (Thermo Fisher Scientific PA5-34895), and goat anti-rabbit conjugated to horseradish peroxidase (Abcam, ab6721). Membranes were imaged using an Amersham Imager 600 (GE Healthcare) using Clarity Western ECL Substrate (Bio-Rad). The intensity of protein bands was quantified using ImageJ and standardized using total (phosphorylated + non-phosphorylated) protein content. Data are representative of three independent experiments.