Asic1a

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).

Protein samples from different regions of mouse brain, including insular cortex and other areas, were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride filters. The filters were incubated overnight at 4 °C with appropriate antibodies. Secondary antibodies conjugated to horseradish peroxidase were added to the filters and then visualized in ECL solution. The visualization was performed via the ImageQuant LAS 4000 mini Molecular Imaging System (GE Healthcare Life Sciences), and the Image J software (NIH) was used for the analysis of band intensity. Antibodies used were as follows: ASIC1a (1:500; Santa Cruz, sc-13905), β-actin (1:1,000; Chemicon, Cat # MAB1501), GAPDH (1:1,000; KangChen, Cat # KC-5G4), GluA1 (1:1,000; Epitomics, Cat # 3861-1), GluA2 (1:1,000; Epitomics, Cat # 3520-1), GluN1 (1:1,000; R&D Systems, Cat # PPS011B), GluN2A (1:1,000; Millipore, Cat # 07-632), GluN2B (1:1,000; Millipore, Cat # MAB5220), PSD-95 (1:1,000; Epitomics, Cat # 2366-1), pGSK3β-Ser9/pGSK3α-Ser21 (1:1,000; Cell Signaling Technology, Cat # 8566) and GSK3β (1:1,000; Cell Signaling Technology, Cat # 12456).