Na931 100ul

Frozen 9 dpf larvae were homogenized and sonicated in 4× Laemmli sample buffer (Bio-Rad, Hercules, CA, USA). Samples were quantified using Pierce 660 nm Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Twenty-five micrograms of protein per sample were separated by SDS-PAGE and transferred onto a PVDF membrane. The PVDF membrane was blocked with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Ak1 polyclonal primary antibody (1:500) (14978-1-AP; Proteintech, Rosemont, IL, USA) was dissolved in 5% skim milk in TBST and incubated for 1 h at room temperature. After washing the membrane with TBST, the membranes were incubated with secondary rabbit antibody (1:1000) (NA934-100UL; GE Healthcare, Chicago, IL, USA) dissolved in 5% skim milk in TBST for 1 h at room temperature. The membrane was washed with TBST and incubated with ECL prime (GE Healthcare) for 5 min at room temperature and imaged on a LuminoGraph II EM (ATTO, Tokyo, Japan). After eliminating signals using hydrogen peroxide, the membrane was treated with FLAG (DDDDK) tag primary monoclonal antibody (1:1000) (M185-3S; MBL, Nagoya, Japan) followed by secondary mouse antibody (1:1000) (NA931-100UL; GE Healthcare).