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Fig. 3 | Journal of Neuroinflammation

Fig. 3

From: CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and l-type Ca2+ channels upstream of p38 MAPK

Fig. 3

Neurotoxic CXCL12 transiently activates p38 MAPK and JNK in rat cerebrocortical cultures. a Complete neuro-glial cell cultures and neuron-depleted cerebrocortical cell cultures were analyzed by Western blotting for the presence of active p38 MAPK and JNK. To obtain glial cells, neurons were depleted by treating cerebrocortical cultures with 300 μM NMDA for 20 min 2 days prior to the preparation of cell lysates. Equal amounts of cellular protein (30 μg) were separated by SDS-PAGE and analyzed by immunoblotting for the indicated proteins. A representative Western blot from one of the three independent neuron depletion experiments is shown. b Cerebrocortical cultures were incubated with recombinant CXCL12 (20 nM) for the indicated time periods, prior to cell lysis on ice. Equal amounts of cellular protein (100 μg) were used for the performance of immunocomplex kinase assays as described in the “Methods” section. The Western blots show representative samples of phosphorylated indicator substrates observed with samples of the 12 and 24 h time points. Kinase activity in CXCL12-exposed samples and vehicle-treated controls was measured for each time point. Vehicle controls were defined as the 100 % baseline value. Note the difference in the dimension of time on the split X-axis. Each time point represents four to seven assessments in duplicate or triplicate in independent experiments. *P ≤ 0.05 compared to control by student’s t test. ‘pp38’/‘p-p38’ and ‘p-JNK’ indicate phosphorylated p38 MAPK and JNK, respectively

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