Actn05

Worm protein samples were prepared by boiling 10 μl pellets of mixed-stage worms in SDS-mercaptoethanol solution. We detected PTRN-1 using antibody OD208A (Wang et al., 2015 (link)); we used the anti-actin antibody ACTN05 (Abcam, Cambridge, MA, C4) as a loading control. We used HRP-linked anti-rabbit IgG NA934V and anti-mouse NXA931 as secondary antibodies (GE Healthcare Lifesciences) at 1:1000 dilution in TBS, and added SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Waltham, MA). The blot result was replicated once.
To test co-immunoprecipitation, a 1:1 ratio of tagged DAPK-1 and PTRN-1 were co-transfected using Lipofectamine 2000 (Invitrogen) into HEK293 cells growing on poly-D-Lysine-coated plates (Sigma-Aldrich) in Opti-MEM (Thermo Fisher Scientific). After two days, cells were collected in cold PBS and lysed in lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1% NP40, 0.5 mM EDTA, 3 mM MgCl₂) for 30 min at 4°C. M2-FLAG conjugated magnetic beads (Sigma) were used for IP, and mouse anti FLAG (F1807, Sigma, 1:1000) and anti HA (HA-7, Sigma, 1:5000) antibodies were used for western blotting. The co-IP was repeated twice.

For detection of protein expression, worms and tissue samples of mice were homogenized in liquid nitrogen. Then the homogenate was lysed on ice for 60 minutes in RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China). The lysates of total protein were loaded 30–50 μg per well and separated on a 10% SDS polyacrylamide gel. Proteins were then transferred to immobilon-PSQ transfer PVDF membrane (Millipore, Bedford, MA). The anti-phospho-YAP-1(Ser104) and anti-YAP-1 rabbit polyclonal antibodies were raised against peptides PIHTRQVpSAPNLH and KHQENSQQDKNQFSVH, respectively, which were obtained from Wuxi AppTec Corporation (Shanghai, China). anti-GFP (#M20004, mouse mAb, 1:1000 dilution; Abmart Inc. Shanghai, China), anti-phospho-Lats1 (Thr1079) (#9113, rabbit polyclonal antibody, 1:1000 dilution; Cell Signaling Technology) and anti-actin antibodies (ACTN05, mouse mAb, 1:1000 dilution; Abcam, Shanghai, China). The secondary antibodies used were HRP-conjugated anti-mouse (1:5000 dilution; Beijing TransGen Biotech Co., China) or rabbit IgG (1:5000 dilution; Abmart Inc.). An imaging system (Amersham Imager 600) was used for documentation of the western results. Protein intensity was analyzed using the ImageJ software (NIH). Uncropped images of Western blots are shown in S11 Fig and S12 Fig.