Ap160p

The protein samples were separated by SDS-PAGE and then transferred to nitrocellulose membrane (PVDF Western Blotting Membranes, Roche) by semidry immunoblotting (Bio-Rad). The presence of proteins on the membrane was confirmed by Ponceau S (Sigma-Aldrich, St. Louis, MI, USA) labeling. After blocking with PBST buffer (PBS, 0.1% Tween 20) containing 3% nonfat milk, the following primary antibodies were used: the antiyeast methionine synthase (MET6; 1:500, X-P05694-N, Abmart, Berkeley Heights, NJ, USA) and the antiyeast alcohol dehydrogenase (1:5000, ab34680, Abcam). The respective proteins were detected after incubation with the horseradish peroxidase-conjugated secondary antibodies (1:10,000, AP160P, Millipore, Merck, and 1:10,000, 111,035,003, Jackson ImmunoResearch, respectively) with a SuperSignal West PICO Chemiluminescent Substrate (Pierce Biotechnology, Waltham, MA, USA), according to the manufacturer’s protocol. The images were captured using an Azure c300 Imaging Systems.

Samples were separated on 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane with Trans-blot Turbo Transfer System (Bio-Rad). Nonspecific binding sites were blocked with 3% nonfat milk in Tris buffered saline containing 0.5% Tween-20. During immunodetection, the membrane was incubated with anti-His antibody (Sigma-Aldrich mouse monoclonal IgG SAB1305538, dilution 1:3000) for 1.5 h followed by horseradish peroxidase–conjugated rabbit anti-mouse secondary antibody (Millipore AP160P, dilution 1:10,000) for 45 min. The immunoreactive bands were detected by Luminata Crescendo Western HRP Substrate (Merck) and imaged with the Multi-Genius Bio imaging System (Syngene).

All drugs and reagents were from Sigma unless otherwise indicated. The main reagents were as follows: Neurobasal medium (Gibco), B27 Supplement (Gibco), Sulfo-NHS-LC-Biotin (Thermo Scientific), NeutrAvidin Agarose Resins (Thermo Scientific), recombinant human BDNF (R&D System), and K252a (Tocris).
The antibodies used for western blotting were ASIC1a (1:500, sc-13905, Santa Cruz) and GAPDH (1:2000, KC-5G4, KangChen). The HRP-conjugated secondary antibodies used for western blotting were goat anti-rabbit IgG (1:2000, AP132P, Millipore), rabbit anti-mouse IgG (1:2000, AP160P, Millipore), and rabbit anti-goat IgG (1:2000, AP106P, Millipore). The primary antibodies used for immunostaining were GFP (1:1000, A10262, Invitrogen), HA (1:1000, 901501, BioLegend), DsRed (1:1000, 632496, Clontech), and PSD-95 (1:1000, ab13552, Abcam). The secondary antibodies used for immunostaining were donkey anti-human IgG DyLight 550 (1:1000, SA5-10127, ThermoFisher), donkey anti-human IgG DyLight 488 (1:1000, SA5-10126, ThermoFisher), donkey anti-mouse IgG Alexa 647 (1:1000, 715-605-150, Jackson ImmunoResearch), donkey anti-rabbit IgG Alexa 568 (1:1000, A10042, Invitrogen), and goat anti-chicken IgG Alexa 488 (1:1000, A11039, Invitrogen).

Tumor tissues were formalin-fixed, paraffin-embedded, and stained using anti-RAS (G12S) mouse monoclonal antibody (NewEast Biosciences, 26186), followed by incubation with the HRP-conjugated corresponding secondary antibody (Millipore, AP160P). The expression levels were evaluated by the H-score method. Scoring was independently reviewed in parallel by two experienced pathologists.

Following protein separation by SDS-PAGE, the gel was incubated in renaturation buffer (0.1 mM DTT, 10 mM Tris (pH 7.5), 20 mM EDTA, 50 mM glycine, 4 M urea) for 10 min, and then for 10 min in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). Then, the gel was placed in a “transfer sandwich” (pad–filter paper–gel–nitrocellulose membrane–filter paper–pad) in a transfer buffer. The transfer was performed for 1 h at 70 V. Next, the membrane was incubated in a TBS-Casein buffer (Tris-Buffered Saline with 1% (w/v) Casein buffer, Bio-Rad Laboratories, Hercules, CA USA) overnight at room temperature. The membrane was transferred into 5 mL of TBS-Casein buffer with 1μg/mL of primary antibodies specific to the c-myc epitope (PSM103-100, SCI-STORE, Moscow, Russia) and incubated for 1.5 h at room temperature. After incubation, the membrane was washed with TBS-Casein buffer for 5 min three times and incubated in 5 mL of TBS-Casein buffer with rabbit anti-mouse antibodies conjugated with horseradish peroxidase (AP160P, SigmaAldrich, Burlington, MA USA) for 1 h. Finally, the membrane was washed in 10 mM tris-HCl (pH 8.0) for 5 min three times. For membrane staining, the reaction of 3,3′-diaminobenzidine with 3% hydrogen peroxide was performed in 10 mL of 10 mM tris-HCl.