Brain tissue from all four groups and regions containing the visual cortex were collected. The mouse visual cortex was dissected and rapidly frozen in liquid nitrogen. Based on our previous work,
18,19 frozen samples were thawed and homogenized in Buffer A (50-mM Tris-Cl, pH 7.5; 1-mM EGTA; 2 mM-EDTA; 100-µM sodium vanadate; 50-nM okadaic acid; 50-mM potassium fluoride; 5-mM sodium pyrophosphate; and 5 µg/µL each of pepstatin A, chymostatin, leupeptin, and aprotinin). Homogenates were then centrifuged at 100,000
g for 30 minutes at 4°C, and the supernatants were collected as the cytosolic fraction. The pellets were resuspended, sonicated, and completely dissolved in Buffer C (Buffer A containing 2% SDS
20) as the membrane fraction. The cytosolic and membrane fractions were used to investigate membrane translocation of cPKCγ, and the membrane fractions were used to analyze phosphorylated GluR1 (pGluR1) at Ser 831 levels. Protein concentrations were determined using a bicinchoninic acid kit (Pierce Biotechnology, Rockford, IL, USA). Albumin dissolved in Buffer A or C at various concentration was used as the standard.
Protein samples (10 µg/lane) were separated using 10% SDS-PAGE, and the proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (0.22 µm; GE Healthcare, Chicago, IL, USA). The transferred PVDF membrane was blocked in 10% no-fat milk in A Tween-20 (Sigma-Aldrich)/Tris-buffered salt solution (TTBS; 20-mM Tris-Cl, pH7.5; 0.15-M NaCl; and 0.05% Tween-20) for 1 hour at room temperature. After washing in TTBS three times for 10 minutes each, the membranes were incubated overnight at 4°C in primary antibodies such as anti-cPKCγ (sc-211, 1:1000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-phospho-GluR1 (Ser831) (ab109464, 1:1000; Abcam), anti-GluR1 (ab109450, 1:1000; Abcam), anti-β-actin (60008-1-Ig, 1:10000; ProteinTech Group, Rosemont, IL, USA), and anti-Na-K-ATPase (ab76020, 1:1000; Abcam). Membranes were then rinsed with TTBS three times (10 minutes each), and incubated in goat anti-rabbit or anti-mouse IgG secondaries at 1:5000 dilutions for 1 hour at room temperature. After rinsing once more with TTBS, protein signal was detected using an enhanced chemiluminescent reagent solution (chemiluminescent horseradish peroxidase substrate; MilliporeSigma, Burlington, MA, USA) and Fusion FX (Vilber Lourmat, Marne-la-Vallée, France).
Quantitative analysis of immunoblots was performed using Fusion Capt 16.15 software (Fusion FX6 XT; Vilber Lourmat). The ratio of the band density of membrane to the band density of the corresponding Na+-K+-ATPase and the ratio of the band density of cytosol to the band density of the corresponding β-actin were calculated. Finally, cPKCγ membrane translocation levels were expressed as the ratio of band density in the membrane fraction to the band densities in both cytosolic and membrane fractions. The pGluR1 (Ser831) levels were expressed as the ratio of the band density in phospho-GluR1 (Ser831) to band density in GluR1.