Activation of Erk in response to various hypothermic conditions and in different cell types. (a) Rat2 cells were left untreated, stimulated with EGF at 37°C, or incubated on ice for various amounts of time followed by warming to 37°C for 12 minutes. When indicated, cells were given a pretreatment of the Mek inhibitor PD098059 (50 μM) for 90 minutes at 37°C. Erk activation was assessed using the mobility shift assay. The positions of inactive p42 and active pp42 Erk are indicated on the right. (b) Rat2 cells were exposed to various temperatures for 4 hours and then warmed to 37°C for 5 minutes. Top: Erk activation was determined with an immunoblot method that uses a primary antibody that recognizes the dually phosphorylated active forms of Erk. Bottom: Equivalence of protein loading was demonstrated by immunoblotting a parallel blot with an antibody that recognizes total Erk. The positions of doubly phosphorylated pp42 and pp44 Erk and total p42 and p44 Erk are shown on the right. (c) Rat2 cells, MDCK cells (canine kidney epithelial cells), KD cells (human primary fibroblasts), CEF (primary chicken embryo fibroblasts), and REC (primary rat embryo fibroblasts) were treated as described below, and Erk activation was assessed using the immunoblotting assay described in b. A, untreated; B, acute growth factor treatment for 5 minutes at 37°C; C, incubation on ice for 4 hours followed by warming to 37°C for 5 minutes; D, incubation on ice for 30 minutes followed by acute growth factor treatment on ice for 5 minutes. Acute growth factor stimulation was EGF (100 ng/mL) for all cell types except CEFs, which were stimulated with 10% FBS. (d) RECs and rat2 cells were left untreated, stimulated with EGF, or incubated on ice for 4, 16, or 24 hours and then warmed to 37°C for 10 minutes. When indicated, cells were given a pretreatment of the Mek inhibitor PD098059 (50 μM) for 90 minutes at 37°C. Erk activation was assessed using a mobility shift assay. Note that only in a and d were SDS-PAGE conditions designed to resolve phosphorylated and nonphosphorylated forms of Erk.