Figure 6

Cytoplasmic characterization of p38α during C2C12 cell differentiation. (A) C2C12 cells were fractionated over a differentiation time course. C, control whole-cell lysate; P, proliferating lysate. Western blotting for phospho-p38 shows a clear increase in the cytoplasm as differentiation proceeds. Nuclei were removed in the initial step of fractionation, and lamin A/C was used to demonstrate that there were no contaminating nuclei in cytoplasmic extracts. Quantification of phospho-p38 expression is shown in Additional file 1 Figure S4. (B) After 48 hours of differentiation, C2C12 cells were lysed and the cytoplasmic fraction was collected and further fractionated into cytosolic (including the cytoskeleton) and noncytosolic fractions. Nuclear marker: lamin A/C; cytoskeletal marker: α-actinin; mitochondrial marker: COX IV; membrane marker: neural cell adhesion molecule (NCAM); endoplasmic reticulum marker: GRP78. (C) Interconnections between the p38α activation pathway and newly discovered in vivo substrates. Proteins previously associated with p38α in its activation pathway are indicated as blue nodes. New substrates are indicated as red nodes. Edge colouring indicates the type of association between nodes.