Protein a coupled magnetic dynabeads

The cells were collected and lysed with RIPA buffer plus protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany)^{17 (link)}. For the chase assay of protein stability, CHX (100 µg/mL), leupeptin (100 µM), and/or MG132 (20 µM) were used for the indicated times. For immunoprecipitation (IP), cleared lysates were incubated with the indicated antibodies (1 µg) and subsequent Protein A-coupled magnetic Dynabeads (Thermo Fisher Scientific). The samples were subjected to SDS-PAGE, and the resulting bands were transferred onto polyvinylidene difluoride membranes for visualization^{18 (link)}. Primary antibodies used in our study are described in Supplementary Table S2. The secondary antibodies used were horseradish peroxidase-coupled anti-mouse IgG and anti-rabbit IgG (GE Healthcare, Chicago, IL, USA). Antibody binding was detected by enhanced chemiluminescence with hyperfilm ECL or an RGB 600 Imager (GE Healthcare).

Cells were collected and lysed with RIPA buffer plus protease inhibitor cocktail (Roche Applied Science) [2 (link)]. For the chase assay of DR5 protein stability, CHX (100 μg/ml) was introduced for the indicated times. For IP, 10% of the lysate was set aside as the input control [2 (link)]. Cleared lysates were incubated with the indicated antibodies (1 μg) and subsequent Protein A-coupled magnetic Dynabeads (50 μl from a stock solution; Thermo Fisher Scientific). Samples were subjected to standard SDS-PAGE, and the resulting bands were transferred onto polyvinylidene fluoride membrane for visualizing specific proteins [2 (link)].
Primary antibodies used in our study are described as followings: IL13Rα1 (ab79277, Abcam), IL13Rα1 (sc101382, Santa-Cruz Biotech), Trail (27,064, Proteintech), DR5 (ab199357, Abcam), ChoP (15,204, Proteintech), STAT6 (5397, Cell Signaling), phosphor-STAT6 (Tyr641) (5397, Cell Signaling), Bax (50,599, Proteintech), and Bcl (12,789, Proteintech). We used normal rabbit and mouse IgG (Santa Cruz Biotechnology, Inc.) as IP controls. The secondary antibodies used were HRP-coupled anti-mouse IgG and anti-rabbit IgG (GE Healthcare). Antibody binding was detected by enhanced chemiluminescence with hyperfilm ECL or an RGB 600 Imager (GE Healthcare).