Pro 2 wash station

Sample homogenates were diluted to a total protein concentration of 900 μg/ml with sample diluent (Bio-Rad, Hercules, CA). The relative abundance of total protein was determined using a Bio-plex kit containing polystyrene, non-magnetic antibody coated beads specific for Akt, c-Jun, CREB, ERK1/2, JNK, MEK1, and p38. Phosphoproteins were assayed using the Bio-plex phosphoprotein kit containing polystyrene, non-magnetic antibody coated beads specific for the following targets phosphorylated at the indicated amino acid residues: Akt (Ser472), c-Jun (Ser63), CREB (Ser133), ERK1/2 (Thr202/Tyr204, Thr185/Tyr187), JNK (Thr183/Tyr185), MEK1 (Ser217/ Ser221), p38 (Thr180/Tyr182), GSK-3α/ß (Ser21/Ser9), HSP27 (Ser78), IκBα (Ser536), p70S6K (Thr421/Ser424), and STAT3 (Tyr705). All beads were analyzed using the Bio-Plex 200 suspension array system, along with the Pro II Wash Station (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions.

To standardize samples for total protein content, tissue homogenates were individually diluted to a total protein concentration of 900 μg/mL with cell lysis buffer (Bio-Rad). Cytokine quantification was performed using a magnetic bead-based multiplex Rat Cytokine Th1/Th2 Kit (Bio-Rad) and measured using a Bio-Plex 200 suspension array system and Pro II Wash Station (Bio-Rad), according to the manufacturer’s instructions. The Th1/Th2 kit included the following cytokines: IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, GM-CSF, IFN-γ, and TNF-α. Results for these 11 cytokines were included in this analysis.