Western blot analysis of p38 MAPK Control Cell extracts using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb
#4511 (upper) and p38 MAPK Antibody #9212 (lower).
Boil for 3 minutes prior to use. Load 15 µl of phosphorylated and nonphosphorylated p38 MAP Kinase Control Cell Extracts per lane.
Supplied in SDS Sample Buffer: 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v phenol red or bromophenol blue. Store at –20°C or at –80°C for long term storage.
Nonphosphorylated p38 MAPK Control Cell Extracts: Total extracts from C-6 glioma cells to serve as a negative control. Supplied in SDS Sample Buffer.
Phosphorylated p38 MAPK Control Cell Extracts: Total extracts from C-6 glioma cells treated with Anisomycin #2222 at 25 ug/ml for 30 minutes to serve as a positive control. Supplied in SDS Sample Buffer.
p38 MAP kinase (MAPK), also called RK (1) or CSBP (2), is the mammalian orthologue of the yeast HOG kinase that participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAPK, p38α, β, γ (also known as Erk6 or SAPK3), and δ (also known as SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAPK is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide (LPS), UV light, and growth factors (1-5). MKK3, MKK6, and SEK activate p38 MAPK by phosphorylation at Thr180 and Tyr182. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 (3) and to phosphorylate the transcription factors ATF-2 (5), Max (6), and MEF2 (5-8). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole) is a selective inhibitor of p38 MAPK. This compound inhibits the activation of MAPKAPK-2 by p38 MAPK and subsequent phosphorylation of HSP27 (9). SB203580 inhibits p38 MAPK catalytic activity by binding to the ATP-binding pocket, but does not inhibit phosphorylation of p38 MAPK by upstream kinases (10).
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