Author: Li, Dongbo; Ji, Hongfei; Niu, Xingjian; Yin, Lei; Wang, Yiran; Gu, Yucui; Wang, Jinlu; Zhou, Xiaoping; Zhang, Han; Zhang, Qingyuan
Title: Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer Document date: 2019_12_19
ID: yj9mb88g_16
Snippet: Cells were lysed in RIPA buffer. Protein samples were sonicated followed by centrifugation at 17 153 g for 15 minutes. Supernatants were collected and protein concentrations were determined using the Bradford Assay (Bio-Rad). Protein lysates were then subjected to 4%-20% Tris-glycine SDS PAGE, and then transferred onto PVDF membranes according to the manufacturer's instructions (Invitrogen). The membranes were blocked in 5% milk-Tris-buffered sal.....
Document: Cells were lysed in RIPA buffer. Protein samples were sonicated followed by centrifugation at 17 153 g for 15 minutes. Supernatants were collected and protein concentrations were determined using the Bradford Assay (Bio-Rad). Protein lysates were then subjected to 4%-20% Tris-glycine SDS PAGE, and then transferred onto PVDF membranes according to the manufacturer's instructions (Invitrogen). The membranes were blocked in 5% milk-Tris-buffered saline with 0.1% Tween (TBS-T) at 23°C for 1 hour, followed by incubation with primary antibodies at 4°C overnight. On the following day, the membranes were washed with TBS-T thrice before incubation with HRP-conjugated secondary antibodies (Cell Signaling) at 23°C for 1 hour. Protein expression was visualized by ECL chemiluminescence (Promega) and quantified using Image J software (National Cancer Institute).
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