Glutamate-dependent activation of Erk1/2 is blocked by B355252 in HT-22 cells . Cultured cells were treated with glutamate 10 h following a 2 h pretreatment with B355252. Cellular fractions (cytosolic and nuclear) were prepared and subjected to immunoblot analysis with total Erk1/2 or pERK1/2 specific antibodies. Membranes were reprobed with GAPDH and H3 histone specific antibodies as controls for protein loading. (A). Cytoplasmic fraction probed with total Erk1/2 (top), pERK1/2 (middle), and GAPDH (bottom) specific antibodies. Erk activation by glutamate was significantly attenuated by pretreatment of cells with B355252 (8 μM). B355252 alone had no effect on phospho-Erk1/2. The bar graph in figure A shows the relative level of active Erk in the samples. ***p<0.001 compared to glutamate-treated cells. (B) Nuclear fractions of samples probed with total Erk1/2 (top), pERK1/2 (middle), and H3 histone (bottom) specific antibodies. +/− indicates presence or absence of compounds.