Figure 2. Synaptic NMDAR-dependent calpain activation degrades PHLPP1 and activates Akt and ERK1/2 pathway. A, Treatment of rat brain P2 fraction aliquots with purified μ-calpain (4 μg/ml) in the presence of 200 μm Ca2+ or purified m-calpain (4 μg/ml) in the presence of 2 mm Ca2+ for 1 h reduced the levels of full-length PHLPP1α and PHLPP1β, as compared with treatment with μ-calpain or m-calpain alone. B, Western blots for rat acute hippocampal slices treated with 20 μm Bic and 100 μm 4-AP for 30 min. In another group, 10 μm CI-III was applied 20 min before Bic and 4-AP treatment. PHLPP1α, PHLPP1β, phospho-Akt S473 (pAkt), Akt, phospho-ERK 1/2 (pERK 1/2), and ERK1/2 were detected by Western blot. C, Quantitative analysis of Western blots similar to those shown in B. The ratios of PHLPP1 to Akt (loading control), p-Akt to Akt, and p-ERK 1/2 to ERK 1/2 were compared. All ratios were normalized to control values before analysis. Bic and 4-AP treatment decreased PHLPP1/Akt and increased p-Akt/ Akt and p-ERK/ ERK. Preapplication of CI-III blocked those changes. *p < 0.05; ns, not significantly different; one-way ANOVA followed by Bonferroni test; n = 3. D, Cultured cortical neurons (DIV12) were treated with 20 μm Bic and 100 μm 4-AP for 30 min. In another group, 10 μm CI-III was applied 10 min before Bic and 4-AP treatment. The levels of PHLPP1, p-Akt, Akt, p-ERK1/2, ERK1/2, and PTEN were detected by Western blot. E, Quantitative analysis of Western blots similar to those shown in D. Bic and 4-AP treatment decreased PHLPP1/Akt and increased p-Akt/Akt and p-ERK/ERK, effects which were blocked by CI-III. PTEN levels were not affected by treatments. *p < 0.05; ns, not significantly different; one-way ANOVA followed by Bonferroni test; n = 3. Error bars indicate SEM.