Phospho p38 kinase

L. plantarum K8 (KCTC 10887BP) was obtained from the Korean Collection for Type Culture (Daejeon, South Korea). Poly I:C was purchased from InvivoGen (San Diego, CA, United States). Pam2CSK4 was purchased from EMC Microcollection GmbH (Tübingen, Germany). Antibodies specific to p38 kinase, phospho-p38 kinase, ERK, phospho-ERK, IκBα, and β-actin were obtained from Cell Signaling Technology (Beverly, MA, United States).

IPEC-J2 cells were seeded in 6-well culture plates and grown until fully confluent. After simultaneous treatment with indicated stimuli for 30 min, the cells were lysed in a lysis buffer. Equal amounts of protein were resolved on 10% SDS-PAGE gels and electro-transferred onto PVDF membranes (Millipore, Bedford, MA, United States). After blocking with 5% bovine serum albumin, membranes were probed with primary antibodies specific for p38 kinase, phospho-p38 kinase, ERK, phospho-ERK, IκBα, or β-actin (Cell Signaling Technology). After washing, membranes were incubated with HRP-conjugated anti-rabbit IgG as secondary antibody. Immunoreactive bands were detected using a luminescent image analyzer (Fuji Film, Tokyo, Japan).

Immunoblot analysis and cell sample harvesting were performed as presented previously [40 (link)]. Briefly, ES cells and/or EBs were washed twice with PBS (pH 7.2) and lysed in sodium dodecyl sulphate (SDS) lysis buffer (50 mM Tris-HCl, pH 7.5; 1% SDS; 10% glycerol). Protein concentrations were determined using the DC protein assay kit (Bio-Rad, USA). Lysates were supplemented with bromophenol blue (0.01%) and β-mercaptoethanol (1%) and incubated for 5 min at 95°C. Equal amounts of total protein (10 μg) were subjected to 8 or 10% SDS-PAGE. After being electrotransferred onto polyvinylidene difluoride membrane (Immobilon-P, Millipore, USA), proteins were immunodetected using appropriate primary and secondary antibodies and visualized by ECL Plus reagent (Amersham Pharmacia Biotech, USA) according to manufacturer's instructions. We used the following primary antibodies: rabbit polyclonal antibodies against p38alpha kinase, Oct4 (Santa Cruz Biotechnology, USA), phospho-p38 kinase, and GAPDH (Cell Signaling Technology, USA). After immunodetection, each membrane was stained by Amido black to confirm equal protein loading. The total level of β-actin was detected as loading control.