Fig. 6.

Stress-induced serine-59 phosphorylation is necessary for CRYAB-dependent IL-6 production by astrocytes. (A–D) Immunocytochemical staining for phospho-Ser-59 (A and B, red) or phospho-Ser-45 (C and D, red) and unphosphorylated CRYAB (A–D, blue) of primary astrocytes either untreated (A and C) or treated with LPS (B and D; 1,000 ng/mL, 24 h) reveals that phosphorylation of Ser-59, localized in the cytoplasm, is enhanced upon stress, whereas Ser-45 phosphorylation, localized in the nucleus, is not affected. (E) ELISA analysis of IL-6 secretion by primary astrocyte cultures treated with inhibitors of p38 (SB203580; 10 μM) and ERK (PD98059; 100 μM), shows that inhibition of p38 and, to a lesser extent, ERK reduces constitutive and stress-induced secretion of IL-6 (1,000 ng/mL LPS; 24 h), depicted as percentage of unstimulated control values. Values depict mean + SEM of four independent experiments and P values determined by unpaired t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).