Mouse anti actr 1

For analyzing the interaction between GDF15 and ALK receptors, co-immunoprecipitation was performed in hPdLFs cultured to 75% confluency in 6-well plates, which were stimulated with 5 ng/mL rhGDF15 for 24 h. Cells were harvested in ice-cold phosphate-buffered saline (PBS) and with centrifugation. Co-immunoprecipitation was performed as previously described [81 (link)]. The following primary antibodies from Santa Cruz Biotechnology (Dallas, TX, USA) were used for precipitation: rat anti-ALK1 (sc-101556), mouse-anti-ACTR-I (ALK2; sc-374523), and rat anti-TGFβ-RI (ALK5; sc-101574). A GDF15-specific antibody (Abcam, Cambridge, UK) was used together with whole cell lysates as positive controls. Prior to protein detection using a semi-dry Western blot, the samples were stored at −20 °C.

For analyzing protein levels, either the samples from the co-immunoprecipitation or samples with ice-cold phosphate-buffered saline (PBS) and centrifugation-isolated hPdLFs were used. Further protein isolation and expression analysis by semi-dry Western blot was performed as previously described [81 (link)]. The following primary antibodies from Santa Cruz Biotechnology (Dallas, TX, USA) were used: rat anti-ALK1 (sc-101556, 1:100), mouse anti-ACTR-I (ALK2; sc-374523, 1:100), and rat anti-TGFβ-RI (ALK5; sc-101574, 1:100). The following secondary antibodies coupled to horseradish peroxidase (HRP) from Thermo Fisher Scientific (Carlsbad, CA, USA) were used: goat anti-mouse IgG HRP (#31430, 1:5000) and goat anti-rat IgG HRP (#31470, 1:5000).