Roti load1 buffer

To quantify actin polymerization in Ishikawa cells, cells were incubated in 100 μl of Triton X-extraction buffer containing 0.3% Triton X-100, 5 mM Tris [pH 7.4], 2 mM EGTA, 300 mM sucrose, 2 μM phalloidin, 1 mM PMSF, 10 μg/ml leupeptin, 20 μg/ml aprotinin, 1 mM sodium orthovanadate, and 50 mM NaF for 5 min on ice. The supernatant containing the soluble proteins was removed by aspiration. The Triton X-insoluble pellet was scraped from the plate directly into 100 μl of RIPA buffer. Any remaining insoluble material was removed by centrifugation. Equal volumes of each fraction were boiled in Roti-Load1 Buffer (Carl Roth, Karlsruhe, Germany) at 100°C for 10 min. Proteins were separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Amersham Biosciences, UK). Non-specific binding sites were blocked by 1 hour at room temperature incubation with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween. The membranes were incubated 2 hours at room temperature with monoclonal rabbit anti-Pan-Actin (D18C11)-HRP conjugated antibody (#12748, 1:1000, Cell Signalling, Frankfurt, Germany). Antibody binding was detected with the Novex ECL Chemiluminescent Substrate Reagen Kit (Invitrogen) and densiometry was performed on scanned images using ImageJ software.

Cytoplasmic and nuclear extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer's instructions. Protein concentration was determined by Bradford assay (Biorad Laboratories) and 20 µg of protein were boiled in Roti-Load1 Buffer (Carl Roth) at 100°C for 5 min. Smad2 phosphorylation in the nuclear and cytoplasmic fractions were further determined by Western blot analysis.