pSMAD inhibition by Western Blot or ELISA

For in vivo measurement of tumor cell pSMAD activity, tumors were harvested from mice at the indicated time points post-treatment, snap frozen in liquid nitrogen, then pulverized in lysis buffer [EDTA 5 mM, EGTA 5 mM, sodium pyrophosphate 30 mM, sodium chloride 6.7 mM, sodium fluoride 50 mM, and 1% triton in water, pH 7.4 with protease and phosphatase inhibitors (Sigma, p8340, p5726, p0044) and sodium orthovanadate 2.5 mM]. Protein concentration was estimated with DC protein assay kit (BioRad).

For in vitro measurements of pSMAD activity, the murine 4T1-LP (luciferase positive) or EMT6-LM2 (EMT6 lung metastasis, passage 2) cells were cultured in 6-well plates in complete media (RPMI with 10% FBS; 0.8 mg/mL G418 selection for 4T1-LP only) until 90% confluent then serum starved overnight in Opti-MEM. Tumor cells were then incubated for 1 hour with galunisertib (starting at 10 μM, reduced by two-fold dilutions to 0.20 μM) in duplicate. The final concentration of DMSO was maintained in each sample at 0.1%. After incubation with the inhibitor, TGFβ1 was added from a 1000-fold stock (10 μg/ml in 40 mM acetic acid, with 0.1% BSA, Invitrogen) to a final concentration of 10 ng/ml to each well, except for the no TGFβ1 control, and the cells were incubated for 45 minutes at 37°C. The reaction was stopped by adding ice cold PBS, then excess TGFβ1 was removed and lysis buffer was added. Next, the samples were homogenized for 15 seconds using Bioruptor^® UCD-200 by Diagenode at medium power. Protein concentration for each lysate was measured using the BCA protein assay kit (Pierce #23225), then protein concentration of the lysate was normalized to 10 mg/mL with lysis buffer.