Anti phospho p38 and anti p38 antibodies

hAF and hNP cells lysates were prepared using RIPA buffer (Biosesang, Seongnam, Korea) with protease inhibitor such as 1 M of PMSF (Roche, BS, Switzerland) and phosphatase inhibitor such as 0.2 mM of Na₃VO₄ (Sigma, MO, USA) and 10 mM of NaF (Sigma). Extracted total protein was evaluated using a bicinchoninic acid (BCA) assay. A sodium dodecyl sulfate (SDS) poly-acrylamide gel (10%) was loaded with 40 µg of total protein, followed by electrophoresis (SDS-PAGE). The resolved proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, CA, USA), which were subsequently blocked with 5% skim milk or 3% BSA and incubated overnight at 4 °C with 5% BSA in TBST (Tris-buffered saline with 0.1% Tween 20) with the primary antibody. Immunolabeling was detected using ECL reagent (Invitrogen, MA, USA). The antibodies used for analysis were anti-phospho-p65, anti-p65, and anti-β-actin antibodies, which were purchased from Santa Cruz Biotechnology (1:1000; Dallas, TX, USA), and anti-phospho-p38 and anti-p38 antibodies were purchased from Cell Signaling Technology (1:1000; Danvers, MA, USA).