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Figure S1. Isoproterenol does not induce ERK1/2 phosphorylation after 2 or 5 min incubation in astrocytes. Bands of 44 and 42 kDa represent p-ERK1 (phosphorylated ERK1) and p-ERK2 (phosphorylated ERK2), respectively (upper rows), or non-phosphorylated ERK1 and ERK2 (lower rows) in primary cultures of astrocytes. Cells were incubated for 2 or 5 min at 0 (Control), 100 nM or 1 µM isoproterenol. (a) Immunoblots from a representative experiment. Similar results were obtained from three independent experiments. Average ERK1/2 phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (b). SEM values are indicated by vertical bars.

Figure S2. ERK1/2 phosphorylation induced by isoproterenol does not require Epac activation in astrocytes. (a) Expression of Epac1 and Epac2 mRNA in brain in vivo and in astrocytes or astrocytes treated with siRNA specific to Epac1/2 for 1 week. TBP is used as a house-keeping gene. The size of the PCR product of Epac1 is 823 bp, Epac2 230 bp and TBP 236 bp. (a-i) Southern blot from a representative experiment. Similar results were obtained from three independent experiments. Average mRNA expression was quantitated as ratios between Epac1or Epac2 and TBP (a-ii and a-iii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from brain sample for Epac1, and #statistically significant (< 0.05) difference from control astrocytes for both Epac1 and Epac2. (b, c and d) Astrocytes were treated with either transfection solution without siRNA {siRNA (−) [Control]} or with siRNA specific to Epac1/2 [siRNA (+)]. One week later, cells were incubated for 20 min in the absence of any drug (Control), in the presence of 1 µM 8-pCPT-2′-O-Me-cAMP (CPT), a specific stimulator of Epac (b), or in the presence of 100 nM isoproterenol (c), or in the presence of 1 µM isoproterenol (d). (b-i, c-i and d-i) Immunoblots from representative experiments. Similar results were obtained from four independent experiments. Average ERK phosphorylation was quantitated as ratios between p-ERK1/2 and ERK1/2 (b-ii, c-ii and d-ii). SEM values are indicated by vertical bars. *Indicates statistically significant (< 0.05) difference from other groups.

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