Rabbit anti amot

MCF7 cells were seeded in Nunc Lab-Tec II 16-well glass chamber slides (Thermo Fisher Scientific) and incubated for 48 h before serum starvation followed by BMP stimulation for the indicated time points. Cells were then fixed using 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Subsequently, PLA was performed using Duolink in situ proximity ligation (Sigma-Aldrich), as previously described (Thymiakou and Episkopou, 2011 (link)), using mouse anti-SMAD1 (sc7965; Santa Cruz Biotechnology), mouse anti-YAP/TAZ (sc101199; Santa Cruz Biotechnology), and rabbit anti-AMOT (Bethyl Labs) at a dilution of 1:200. Imaging occurred with an inverted epifluorescence Axiovert 200M microscope (Zeiss). The number of heteromers was quantified using BlobFinder image analysis software as previously described (Allalou and Wahlby, 2009 (link)). At least 500 cells per experimental condition were quantified of each replicate and normalized to starved cells.

Immunoprecipitation of overexpressed proteins from HEK293T cells (24 h posttransfection) and endogenous proteins from MCF7 cells (48 h postseeding) was performed using a modified radio-immunoprecipitation assay buffer (RIPA) freshly supplemented with inhibitors (1 mM PMSF, 2 mM Na₃VO₄, 20 mM Na₄P₂O₇, 50 mM NaF, complete protease inhibitor cocktails [Roche]) as previously described (Benn et al., 2015 (link)). TCLs were taken before incubating the lysates with the indicated primary antibody (1–4 µg/lysate) overnight. rabbit anti-AMOT (TLE) antibody, used for AMOT-BMPR2 interactions, was kindly provided by Lars Holmgren (Karolinska Institutet, Stockholm, Sweden). Furthermore, mouse anti-HA tag (Sigma-Aldrich), rabbit anti-SMAD1 (Cell Signaling Technologies), and rabbit anti-AMOT (BethylLabs) antibodies were used for pull down. Control samples were incubated with either isotype control antibodies or recombinant protein A-Sepharose beads (GE Healthcare). Immunocomplexes were precipitated at 4°C for 2 h with recombinant protein A-Sepharose beads and subsequently washed 3–5× with fresh lysis buffer including inhibitors. Proteins were eluted with 2× Laemmli sample buffer and boiled for 10 min at 95°C prior to Western blotting.