Ab7750

Following treatment with Antimycin A, animals expressing EMB-9::mCherry (n = 300) were collected in 1.5-ml microfuge tubes. After 2 washes with M9 buffer, animals were immediately frozen in liquid N₂. This was followed by thawing and addition of 100 µl of 1-time SDS dye. Next, the tubes were placed in a liquid bath sonicator (Bioruptor; Cosmo Bio, Tokyo, Japan) on 5-s on and off cycles for 10 min, followed by incubation at 95°C for 10 min. Samples were then run on an 8% SDS gel, after which they were transferred onto a PVDF membrane (88518; Thermo Fisher Scientific, Waltham, MA, USA) and blocked in 5% skimmed milk for 1 h. Next, the membrane was incubated overnight at 4°C in primary antibody (anti-mCherry, ab125096, anti-α-tubulin, ab7750; Abcam, Cambridge, MA, USA) prepared in 2.5% milk. This was followed by 1 wash with 1-time PBS with Tween 20 for 5 min, and the membrane was transferred to secondary antibody (sheep anti-mouse; GE Healthcare, Waukesha, WI, USA; NA93IV, po448, goat anti-rabbit, Agilent Technologies, Santa Clara, CA, USA) incubated at room temperature for 3 h. The membrane was washed thrice with 1-time PBS with Tween 20, and the bands were then visualized using a chemifluorescence kit (Western Lightning Plus-ECL; Takara, Kyoto, Japan).