Beyoecl plus substrate system

The activity of rCiDf to promote the cleavage of C3 protein was analyzed according to previous descriptions [23 (link)]. Briefly, 25 µL of rCiDf protein (40 µg/mL) were incubated with the serum of grass carp at 16 °C overnight. Equivalent rGST (40 µg/mL) proteins and PBS incubated with grass carp serum were set as the negative and blank control, respectively. Western blot analysis was conducted for the detection of C3 protein cleavage in grass carp serum. These three sample types were detected by first separating proteins using 10% SDS-PAGE followed by transfer to a polyvinylidene difluoride membrane (Millipore, MA, USA). The membranes were blocked in a QuickBlock Blocking Buffer (Beyotime, Shanghai, China) for 15 min at 25 °C and incubated with the rabbit-anti-grass-carp C3 polyclonal antibody (diluted by 1:1000) overnight. After washing three times with Tris-buffered saline containing 0.05% Tween-20, the membrane was incubated with a 1:2000 dilution of HRP AffiniPure goat anti-rabbit IgG (Abclonal, Wuhan, China) for 1 h at 25 °C. The membranes were finally incubated in the BeyoECL Plus substrate system (Beyotime, Shanghai, China) for imaging under the GeneSys Imaging System (Alcatel, Paris, France). The band intensity was quantified and analyzed using ImageJ software. The β-actin was used as an internal reference protein.

Cytoplasmic and nuclear fraction separation was performed using NE-PER nuclear and cytoplasmic extraction reagents (Thermo, MA, USA). Total cellular proteins were collected using RIPA lysis buffer (Beyotime, Jiangsu, China). Equal amounts of protein were electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A wet transfer method was used to electrophoretically transfer proteins from the native gel to nitrocellulose membranes, which were then probed with anti-p-LATS1/2^T1079/1041 (1:1000, Sigma, SAB4504615), anti-LATS1 (1:1000, Sigma, SAB1300096), anti-p-MST1/2 (1:1000, Sigma, SAB4504042), anti-MST1 (1:1000, Abcam, AB124787, Cambridge, UK), anti-Yap1 (1:1000, Abcam, AB39361), anti-p-Yap^S127 (1:1000, Abcam, AB76252) antibodies, and developed with the BeyoECL Plus substrate system (Beyotime). The blots were stripped and reprobed with GAPDH antibody (1:2000, Bioworld, AP0063, CA, USA) to confirm equal protein loading.

All treated cells were washed with PBS and lysed by RIPA and PMSF at a ratio of 100:1 to obtain the total protein for Western blot. Equal amount of proteins was separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane. After transferring, the polyvinylidene fluoride membrane was blocked in 5% fat-free milk/1 × TBS/0.1% Tween-20 for 1 h at room temperature and then incubated with primary antibodies with gentle agitation overnight at 4°C. And then, the membrane was washed with 1 × TBS/0.1% Tween-20 before incubated with the secondary antibody according to the species of primary antibody for 1 h at room temperature. Immunoblots were performed by using the BeyoECL Plus substrate system (Beyotime), followed by washing with 1 × TBS/0.1% Tween-20. Western blot analyzed of LC3A/B (Abcam ab128025) and GAPDH (AP0063, Bioworld).