Western Blot for PCDH1, β3 Integrin, and DAF

Performed as described (Jangra et al., 2018 (link)). Briefly, cells were lysed in radioimmunoprecipitation assay lysis buffer (RIPA) containing protease inhibitors (Sigma). Cell extracts were normalized by Bradford assay, and 40 µg of protein was added per lane. The extracted proteins were separated by 10% SDS–PAGE and transferred onto 0.22 µm nitrocellulose membranes (GE Healthcare Life Sciences). Blots were cut approximately in half for subsequent incubation with primary antibodies and aligned back together for imaging. Antibodies used were as follows: Mouse α–PCDH1 (sc-81816, Santa Cruz) 1:200; Rabbit-α–β3 Integrin (#4702, Cell Signaling) 1:300; Mouse-α–DAF (NaM16-4D3, Santa Cruz) 1:200; Mouse α–β-Actin (sc-4778, Santa Cruz) 1:300. IRDye 680LT Goat α-Rabbit IgG or IRDye 680LT Goat α-Mouse secondaries Abs (LI-COR) were used at a dilution of 1:10,000, and the final blot was then imaged using a LI-COR Fc fluorescent imager.