Rabbit phospho smad1 5 9 antibody

hFOB spheroids embedded in collagen gels were washed with PBS and fixed with 4% paraformaldehyde for 1 h, washed with PBS, and permeabilized with 0.1% Triton X-100 in PBS overnight at 4°C. Afterward, the samples were blocked with 3% BSA in PBS overnight at 4°C and then incubated with a rabbit phospho-SMAD1/5/9 antibody at 1:400 (D5B10, Cell Signaling) and a mouse anti-YAP antibody at 1:200 (63.7, Santa Cruz Biotech) in 3% BSA overnight at 4°C. Next, the samples were washed 3× with PBS for 30 minutes per wash and incubated with DAPI, Acti-stain 670 phalloidin, and secondary antibodies in 3% BSA overnight at 4°C. The samples were washed 3× with PBS as described above and imaged by spinning disk confocal microscopy.
For image analysis of experiments nuclear/cytoplasmic YAP ratio, background subtraction was performed in ImageJ, and images were subsequently segmented with MATLAB to generate masks for actin and DAPI channels that were used to measure the mean nuclear and cytoplasmic intensity of each region. To quantify YAP, the total nuclear or cytosolic intensity was calculated, and nuclear intensity was averaged over cytosolic intensity to generate a nuclear/cytoplasmic YAP ratio.