Bca concentration detection kit

Organoids were incubated with alpelisib, fulvestrant or a combination for 6 days and then washed with cold 1 × PBS. These cells were suspended in RIPA lysis solution (Biyuntian, P0013C) containing 1% protein and phosphorylation inhibitor (Abmole bioscience, M5293) on ice for 30 min. Then, the cells were centrifuged at 13,000 g at 4°C for 15 min and the supernatants were collected. The concentration was detected by the bicinchoninic acid (BCA) concentration detection kit (Biyuntian, P0012). The remaining parts were mixed in 5× loading buffer (Biyuntian, P0015), boiled for 10 min in a metal water bath, and then subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). After electrophoresis, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated against primary antibody for 24 h. Membranes were washed with tris buffer saline‐Tween (TBS‐T) and incubated with a 1:5000 dilution of secondary antibody for 2 h. Protein bands were then visualized by Ultra Luminol/Enhancer Reagent (New Cell & Molecular, China). The following antibodies were used: PI3K‐110α (Abcam, ab1678, 1:500), p‐AKT (CST, 4060S, 1:1000), AKT (CST, 4691S, 1:1000), p‐mTOR (CST, 5536S, 1:1000), mTOR (CST, 2983S, 1:1000), p‐S6 (CST, 4857S, 1:1000), ER (CST, 13258S, 1:500) and GAPDH (Proteintech, 60,004‐1‐Ig, 1:10000).