Nitrocellulose blotting membrane

Cells were harvested and lysed in a lysis buffer containing 50 mM Tris-Cl pH 7.4, 250 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM NaF and cocktail of protease inhibitors. Following lysate clearance with centrifugation for 30 min at 12,000 rpm and 4 °C, protein quantification was performed with Coomassie Brilliant Blue G250 (Beyotime). Proteins from cellular fractions were separated on SDS-PAGE and transferred onto a 0.45 μm nitrocellulose blotting membrane (Amersham). Membranes were probed with corresponding primary antibodies after blocking with 5% milk in PBS. After extensive washing with PBS/T (0.5% Tween-20 in PBS buffer), the membranes were incubated with IRDye^® 800CW or 680RD secondary antibodies (LI-COR). Fluorescence signals were detected using Odyssey^® CLx imager. Images were acquired using Image Studio 5.0 (LI-COR).

Proteins were loaded on 7.5% or 4 to 20% Mini-PROTEAN TGX Stain-Free Gels (Bio-Rad) after the addition of 4× Laemmli sample buffer and denaturation at 95°C for 5 min. After migration, proteins were transferred to a nitrocellulose blotting membrane (Amersham). Membranes were washed in tris-buffered saline (TBS) and then blocked for 1 hour at room temperature in blocking solution (TBS, 10% milk, and 0.1% Tween 20). The primary antibodies were diluted in washing buffer (TBS, 2% milk, and 0.1% Tween 20) and incubated with the membranes overnight at 4°C at the following concentrations: anti-Ph (1:1000), anti-E(z) (1:1000), anti-Pho [1:1000, a gift from J. Kassis; (44 (link))], anti-GFP (1:1000: Takara, JL-8), and anti-tubulin (1:1000; Sigma-Aldrich, DM1A) (table S4). Membranes were washed three times in washing solution and incubated with the appropriate HRP-conjugated secondary antibodies for 2 hours at room temperature. Detection was performed using the Clarity Western ECL substrate (Bio-Rad) according to the manufacturer’s instructions, and the signals were analyzed with the ChemiDoc MP Imaging System (Bio-Rad).

Cells treated as described were harvested, washed with PBS and lysed in 1x LDS buffer (Life Technologies, Carlsbad, US). Proteins were separated using a 4–12% NuPAGE Bis-Tris gel (Life, Technologies, Carlsbad, US) and transferred to a nitrocellulose blotting membrane (Amersham, Little Chalfont, UK) by electroblotting. Target proteins were detected with the help of the WesternBreeze Chemiluminescent Kit, according to the manufacturer’s instructions.

The proteins were extracted from fresh brain tissues (ACC and RSC) from postnatal 16 mice after decapitation. The extraction was made with RIPA buffer (Millipore; 20–188) in azote and sonicated later. The total amount of proteins was detected with Pierce BCA protein assay kit (Thermo Scientific; 23225). After denaturalization (at 92 ºC, 10 min with β-mercaptoethanol) 20 µg of proteins of each sample were loaded on SDS–polyacrylamide gel (12%) and electrophoresed followed by wet transfer in nitrocellulose blotting membrane (Amersham; 10600007). The membranes were incubated with primary antibodies overnight at 4 ºC (with TBST and BSA 5%): rabbit GluA2 (1:4000, Fisher; AB1768IMI), mouse anti-GluA1 (1:1000, Rockland, 200-301-D61), rabbit anti-GluK5 (1:1000, Abcam, ab32672), and mouse GAPDH (1:10,000, GeneTex; GTX627408). The membranes were washed with TBST and incubated with the secondary antibodies conjugated with HRP: anti-rabbit (1:4000, Vector; BA-1000) and anti-mouse (1:4000, Vector; BA-9200). The membranes were treated with Immobilon Forte (Millipore; WBLU0100) and exposed in dark room. Images were taken with the Amersham Bioimager 680 and to analyze the ratio of gray values between samples Image J was used. The protein level was expressed in arbitrary units (A.U.) calculated as the quotient between the area of the GluA2 band over the area of the GAPDH band.

Virus-like particle and cell lysate samples were loaded on a 15% SDS polyacrylamide gel followed by transfer to a nitrocellulose blotting membrane (Amersham, Germany). Membranes were blocked for 1 h in 5% non-fat dry milk (Blocking buffer), then incubated with the corresponding primary antibody for 2 h at room temperature. After washing with wash buffer (0.2 M Tris-HCl (pH 7.5), 8.76 g/L NaCl and 0.25% Tween 20), the membranes were incubated with the appropriate HRP-conjugated secondary antibodies for 2 h at room temperature. Membranes were washed with wash buffer three times for 10 min each and subsequently imaged on an image analyzer LAS-3000 (Fujifilm, Japan) using ECL prime chemiluminescent reagent (GE Healthcare, Italy) according to the manufacturer’s instructions. The representative data of immunoblots repeated at least four times are shown in each figure.