Ipvh00010

Worms were collected, washed several times with PBS, and stored at −80°C until analysis. Frozen worms were homogenized with RIPA buffer containing protease inhibitor cocktail. Protein concentrations were determined using DC protein assay (Bio‐Rad). Protein (20–50 µg) from each sample was resolved in a 12% SDS‐PAGE gel, transferred to a PVDF membrane (Millipore, IPVH00010), blotted with the antibody against GFP (Abcam) in 5% nonfat milk at 4°C overnight, and detected with an ECL system (Amersham). Coomassie blue staining of the blot was used to assess loading of protein samples.

RIPA lysate was used to prepare tissue homogenate to extract protein and NP-40 lysate to obtain cell protein. The protein concentration was measured using the BCA protein kit (TaKaRa, Otsu, Japan, T9300A). Protein was degenerated in sodium dodecyl sulfate (SDS) buffer, followed by separating on SDS-polyacrylamide electrophoresis (PAGE) gel using electrophoresis. After that, they were transferred onto polyvinylidene difluoride (PVDF, Millipore, USA, IPVH00010) membrane and incubated with primary antibody actin (Abcam, USA, ab8226, 1 : 400), BAX (Abcam, USA, ab32503, 1 : 200), Bcl-2 (Abcam, USA, ab182858, 1 : 200), caspase-3 (Abcam, USA, ab32351, 1 : 1000), Cbfα-1 (Abcam, USA, ab113203,1 : 200), OPN (Abcam, USA, ab228748, 1 : 200), SM-α (Abcam, USA, ab7817, 1 : 500), Akt (Abcam, USA, ab8805, 1 : 200), p-Akt (Abcam, USA, ab38449, 1 : 100), ERK1/2 (Abcam, USA, ab184699,1 : 1000), p-ERK1/2 (Abcam, USA, ab278538,1 : 1000), and β-actin (Abcam, USA, ab8226, 1 : 1000) overnight at 4°C. Next, they were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies IgG (Abcam, USA, ab150077, goat anti-rabbit, 1 : 5000) at room temperature for 45 min. After the membrane was washed in TBST, the blot was visualized by the enhanced chemiluminescence ECL kit (Thermo Fisher Scientific, NY, USA).