Polyclonal rabbit phospho p44 42 mapk antibody

Western blots were performed as described previously with some modification (Xi et al., 1999 (link)). Astrocytes were collected and immersed in Western sample buffer (62.5 mM Tris-HCl, pH 6.8, 2.3% sodium dodecylsulfate, 10% glycerol, and 5% β-mercaptoethanol) and were then sonicated for 10 seconds. Protein concentrations were measured using Thermo Scientific Pierce BCA Protein Assay Kit. Samples were loaded after 5 minutes boiling at 95 C. The protein was transferred to a hybond-C pure nitrocellulose membrane (Amersham). The membranes were blocked in 5% Carnation non-fat dry milk for 2 hours at room temperature. Blots were washed and membranes were incubated with the following primary antibodies: polyclonal rabbit phospho-p44/42 MAPK antibody (1:2000; Cell Signaling, MA, U.S.A.), polyclonal rabbit p44/42 MAPK antibody (1:2000; Cell Signaling), polyclonal rabbit phospho-p90RSK (T359/S363) antibody (1:1000; Cell Signaling), polyclonal rabbit p90RSK antibody (1:1000; Cell Signaling) and polyclonal rabbit HSP27 antibody (1:1000; Cell Signaling). The appropriate second antibodies were applied for 1 hour at room temperature. Proteins were visualized with the ECL chemiluminescence system (Amersham) and exposed to Kodak X-OMAT film. The relative densities of the protein bands are analyzed with ImageJ 1.44 software.

Astrocytes were grown on chamber slides and fixed with 4% paraformaldehyde. Slides were rinsed and the cells permeabilized with 100% methanol at −20 C for 5 minutes. Polyclonal rabbit phospho-p44/42 MAPK antibody (1:400), polyclonal rabbit phospho-p90RSK (T359/S363) antibody (1:200) and polyclonal rabbit HSP27 antibody (1:200; Cell Signaling) were applied and the slides incubated overnight at 4 C in a moist chamber after 10% normal goat serum blocking for 1 hour at room temperature. After washing three times, the slides were incubated with rhodamine goat anti-rabbit IgG (1:500, Chemico, Temecula, CA, U.S.A.) for 1 hour at room temperature and astrocytes were then photographed using a fluorescence microscope.