m four experiments. (d) Cultured cortical neurons (∼DIV21) were incubated in the absence (Con) or presence of NMDA (100 µM) for 10 min. Proteins were solubilized using RIPA buffer and I-2 was immunoprecipitated followed by blotting for PP1 and T320 phosphorylation. Bar graph shows data from three experiments. (e) Control, synaptic, or extrasynaptic NMDA receptor stimulations were performed on cultured cortical neurons before the proteins were solubilized using RIPA buffer, and I-2 was immunoprecipitated followed by analysis by blotting for PP1. Bar graph shows data from three experiments. (f) Cultured cortical neurons were infected with recombinant Sindbis virus encoding CFP--I-2 (labeled as I-2(WT)) or CFP--2(T72A) (labeled as I-2(T72A)) for 1 d before the RIPA soluble fractions were used for immunoprecipitation with GFP antibody (GFP IP). Samples were analyzed by blotting for GFP and I-2 phosphorylation status on T72. Note: two different exposures of PP1 and I-2pT72 blots are presented.](JCB_201303035R_Fig3){#fig3}

We initially examined phosphorylation of I-2 at T72 in cortical neurons that expressed CFP-I-2 via recombinant Sindbis virus infection. We observed robust basal I-2 phosphorylation at T72, which was substantially decreased in response to NMDA treatment ([Fig. 3 b](#fig3){ref-type="fig"}; the specificity of the antibody was confirmed in assays shown in [Fig. S3 a](http://www.jcb.org/cgi/content/full/jcb.201303035/DC1){#supp3}), and a similar effect was observed for endogenous I-2 ([Fig. 3 c](#fig3){ref-type="fig"}). PP1 and I-2 form a 1:1 complex. To examine the regulation of PP1 in the PP1--I-2 complex we immunoprecipitated I-2. In response to NMDA application, the level of PP1 phosphorylation at T320 in the PP1--I-2 complex was also decreased ([Fig. 3 d](#fig3){ref-type="fig"}). This suggested that PP1 in the I-2 complex is activated via removal of the pT320 inhibitory phosphorylation, and that this occurred in parallel with I-2 dephosphorylation at T72.

Notably, we observed an increase in bind