Anti ha antibody

Cells were lysed in ice-cold RIPA buffer with protease inhibitors for 10 min on ice, the lysates were then cleared by centrifuging the cells at 15,000× g for 10 min at 4 °C. For IP of endogenous Gsk-3α/β, 500 μg whole cell extract from each sample was incubated with Protein G Sepharose (Cell Signaling Technology) and 5 μg anti-Gsk-3α/β antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4 °C with rotation. After incubation, the beads were washed thrice with RIPA buffer, followed by a final wash in 1× PBS. The immunoprecipitants were resolved by SDS-PAGE and subjected to immunoblotting. For IP of exogenously expressed proteins in HEK293, 2 μg of anti-Flag antibody (Cell Signaling Technology) was used; for HA, 2 μg of anti-HA antibody (Cell Signaling Technology) was used.

W20 cells and HEK293 cells were grown in DMEM containing 10% (v/v) FBS, 50 U/mL penicillin/streptomycin, and 2 mM L-glutamine. These cells were transfected with Myc-ACVR1, Myc-ACVR1[R206H], and HA-ACVRIIB alone or in various combinations. W20 cells were transfected using X-tremeGENE 9 DNA transfection reagent (Sigma-Aldrich, 06 365 787 001) and HEK293 cells were transfected using TransIT-293 DNA transfection (MirusBio, MIR 2700) by following the manufacturers’ protocols. After transfections, cells were incubated overnight in the complete media. The following day, cells were switched to serum-free media (in the presence or absence of ACVRIIB-Fc). Forty-eight hours after transfection, membrane fractions of the transfected cells were isolated using the Mem-PER Plus membrane protein extraction kit (Thermo Fisher Scientific, 89842). Membrane fractions were resuspended in the lysis buffer of the Myc-IP kit (Thermo Fisher Scientific, 88844) and Myc immunoprecipitation was performed using isolated membrane fractions by following the manufacturer’s protocol. Immunoblotting was performed using immunoprecipitation input and elution samples as described above. Anti-ACVR1 antibody (Abcam, ab155981) and anti-HA antibody (Cell Signaling Technology, 3724) were used detect Myc-ACVR1 and HA-ACVRIIB, respectively.

Cells were harvested 24 h after transfection or infection with expression vectors and subjected to SDS-PAGE and Western blotting under the conditions described previously [11 (link)]. A rabbit anti-human REIC/Dkk-3 antibody was raised in our laboratory [11 (link)]. Goat anti-human KLF16 antibody (Abcam, Inc., Cambridge, MA), anti-6x histidine antibody (MBL Co., Nagoya, Japan), anti-HA antibody (Cell Signaling Technology, Danvers, MA), and anti-GFP antibody (Clontech) were purchased.

For crude protein extracts, cells were lysed in RIPA buffer containing 1% NP40 and 0.1% SDS (Carl Roth) supplemented with ‘Complete’ and ‘PhosStop’ protease/phosphatase inhibitor cocktail (Sigma Aldrich) as described^{50 (link)}. Immunodetection of cellular extracts was performed using an anti-KIBRA (Santa Cruz Biotechnology; 1:500), anti-SP1 (Merck; 1:1000), anti-YAP (Santa Cruz Biotechnology; 1:1000), anti-pYAP (Ser127; Cell Signaling; 1:1000), anti-LATS1 (Merck; 1:1000), anti-pLATS1 (Thr1079; Cell Signaling; 1:500) and anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:20000 or Merck; 1:10000). Sample loading was controlled by β-actin detection (Cell Signaling; 1:5000) and anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:10000 or Merck; 1:20000). ZFP226 detection was conducted using anti-HA antibody (Cell Signaling; 1:1000) and anti-mouse secondary antibody (Santa Cruz Biotechnology; 1:20000). Western blots were repeated at least three times and band intensities were quantified using ImageJ^{51 (link)}.