Apc 030

Western blot analysis was performed on Mini-PROTEAN^® TGX™ Gels (Bio-Rad Laboratories). Proteins were electrotransferred onto Immobilon-P PVDF membrane (millipore, pore size 0.45 mm). After blocking 1 h in Tris-HCl-buffered saline with 0.2% Tween-20 (TBST) and 5% non-fat milk (Bioshop^® Canada), the membranes were incubated overnight with primary antibody in TBST containing 2% non-fat milk. The rabbit polyclonal anti-HCN2 antibody (1:500; APC-030, Alomone Labs), rabbit polyclonal anti-HCN4 antibody (1:1000; APC-052, Alomone Labs), mouse monoclonal anti-SGO1 antibody (1:1000; ab58023, Abcam), mouse monoclonal anti-GAPDH antibody (1:1000; 10R-G109a, Fitzgerald), mouse monoclonal anti-GFP antibody (1:1000; MA5-15256, Invitrogen), rat monoclonal anti-mCherry (1:1000; M11217, Invitrogen) and mouse monoclonal anti-PP2A C subunit (1:1000; SAB4200266, Sigma-Aldrich) were utilized to detect HCN2, HCN4, SGO1, GAPDH, GFP, mCherry and PP2A C proteins respectively. Detection was carried out by the use of horseradish peroxidase conjugated secondary antibodies (Donkey anti rabbit secondary antibody, 1:5000, #711035152, Jackson; Donkey anti mouse secondary antibody, 1:5000, #711035151, Jackson; Donkey anti rat secondary antibody, 1:5000, #A18745, Invitrogen), photographic film (Agfa NV) and the Western Lighting^® plus ECL kit (PerkinElmer).

Ventral hippocampal tissues were isolated and homogenized with RIPA lysis buffer (R0010, Solarbio, Beijing, China). Proteins were separated using a 15% SDS-PAGE gel and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (ISEQ00010, Merck Millipore, Burlington, VT, USA). Membranes were then blocked with 5% non-fat milk for 1 h at room temperature, incubated at 4 °C overnight with primary antibody against HCN1 (1:200, APC-056, Alomone labs Jerusalem, Israel), HCN2 (1:200, APC-030, Alomone labs, Jerusalem, Israel) and β-actin (1:2000, TA-09, ZSGB-Bio, Beijing, China), respectively. After washing three times with tris-buffered saline containing 0.1% Tween 20, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000, ZB-2301/ ZB-5305, ZSGB-Bio, Beijing, China) at room temperature for 1 h. Protein bands were visualized using a chemiluminescence imaging system (Tanon-5200) and quantified using ImageJ software (version 1.47v; NIH, Bethesda, MD, USA).