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. Author manuscript; available in PMC: 2021 Jun 14.
Published in final edited form as: Mol Neurobiol. 2020 Mar 25;57(6):2539–2550. doi: 10.1007/s12035-020-01871-z

Fig. 2. Extracellular Mg2+ is necessary for NMDA caused delayed CREB phosphorylation in neurons with Ca2+ chelated in conditioned medium.

Fig. 2

a. Increasing Mg2+ in conditioned medium decreases basal pCREB (1 hour time point). b. pCREB levels after the chelation of Ca2+ alone (with 10 mM EGTA). Neurons were treated for 1 hour before cell harvesting. c. Increasing Mg2+ in conditioned medium did not affect basal pCREB levels when calcium is chelated by EGTA in conditioned medium. 10 mM EGTA was added to conditioned medium for 1 hour before extra MgCl2 was included for 10 or 60 minutes. d-h. Mg2+ dependent CREB phosphorylation in response to NMDA application in neurons. d. NMDA treatment led to increased pCREB in neurons in conditioned medium. e: Delayed CREB phosphorylation was detected after the removal of external Ca2+ with EGTA. (f-g) Increase of Mg2+ concentrations in conditioned medium (with EGTA) enhanced NMDA effects on CREB phosphorylation. h: Further chelating magnesium in conditioned medium blocked the delayed pCREB by NMDA as in (e). i, j. Quantifications of d-h. (**, P<0.01; **, P<0.001)