Zm 0442

A total of 30 pairs of primary BC fresh tissues and cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (strong; Beyotime, China) in the presence of protease inhibitors (Roche). Equal amounts of cellular proteins were loaded into each well and resolved using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. The samples were heated for 5 min at 95°C. Polyvinylidene fluoride membrane blotting was subsequently performed under standard conditions. For immunoblotting, the following primary antibodies were used against pEGFR (ab40815; Abcam, US), EHD1 (ab51504; Abcam, Hong Kong), RAB11FIP3 (ZM-0442; ZSGB-BIO, China), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma). The primary antibody solution was diluted 1:1000 and incubated with the membrane overnight at 4°C. The primary antibody against GAPDH (Sigma) solution was diluted 1:5000 and incubated with the membrane overnight at 4°C. The membrane was subsequently washed three times for 10 min with a mixture of tris-buffered saline and Tween 20 (TBS-T) prior to adding a 1:5000 dilution of the secondary antibody, which was diluted in the TBS-T solution at room temperature. The membrane was then incubated on a swing bed for 1 h prior to three 10-min washes with TBS-T.