ef-type="fig"}).

![**Inhibitor-1 (I-1) and calcineurin are not critical for regulating PP1 activity in cortical neurons.** (a) Cultured cortical neurons were incubated in the absence (Con) or presence of NMDA (20 µM for 10 min) without or with 20 µM cyclosporine A (CSA), 1 µM FK506, or a combination of both CSA and FK506, pre-applied to cultures for 10 min. Proteins were analyzed by SDS-PAGE and immunoblotting with antibodies to phospho-T320 in PP1 (pT320) or total PP1. (b) Cultured cortical neurons were infected with lentivirus encoding ShRNA against I-1 or scrambled ShRNA. 5 d later, the samples was treated without (Con) or with NMDA (20 or 100 µM) for 10 min before the proteins were analyzed by SDS-PAGE and blotted with pT320, PP1, I-1, and I-2 antibodies (I-2 blotting was used to show specificity of I-1 knock-down \[KD\]). Bar graph shows data from three experiments. \*, P \< 0.05; \*\*, P \< 0.01; \*\*\*, P \< 0.001 compared with control. (c) Cultured neurons were incubated without (Con) or with okadaic acid (OA: 10 or 200 nM, or 1 µM), fostriecin (200 nM), FK506/CSA (1 µM /20 µM), Sp-cAMP (100 µM), or Rp-cAMP (100 µM) for 10 min or otherwise specified. Proteins were analyzed by blotting with I-1 and pT35--I-1 antibodies.](JCB_201303035_Fig2){#fig2}

We also assessed the phosphorylation of I-1 at T35, the site phosphorylated by protein kinase A that converts I-1 into a potent PP1 inhibitor. The basal level of I-1 phosphorylation at T35 was low and could be increased approximately twofold by stimulation of protein kinase A with Sp-cAMP ([Fig. 2 c](#fig2){ref-type="fig"}). Moreover, incubation with CSA plus FK506 had little/no effect on I-1 phosphorylation at T35. Interestingly, incubation of neurons with a low dose of okadaic acid (OA; 10 nM, specific for PP2A) or the PP2A-specific inhibitor fostricin, did not significantly affect I-1 phosphorylation at T35. However, higher concentrations of OA (200 nM or 1 µM, which would also inhibit PP1) were able to increase I-1 phosphorylation at T35 by more t