Sab5600064

The tissue lysates were prepared in ice-cold tissue lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS) containing 50 mM NaF, 2 mM Na₃VO₄, protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA) and phosphatase inhibitors (Sigma-Aldrich) and total protein was extracted as previously described^{50 (link)}. Samples from tissue lysates were resolved by SDS-PAGE and then transferred to a nitrocellulose membrane. After 1 h blocking at 4 °C using 5% BSA in phosphate-buffered saline containing 0.1% Tween-20 (PBST), the membrane was incubated overnight with antibodies against phospho-IRβ (sc-25103, Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Akt (SAB5600064, Sigma-Aldrich), and α-tubulin (T9026, Sigma-Aldrich) in PBST at 4 °C. After 3 PBST washes, membranes were incubated with secondary antibody for 1 h at room temperature. Chemiluminescence was performed using a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Cell lysates are prepared using Ripa Buffer and run in SDS-PAGE gel as per standard protocol. After the proteins are transferred in nitrocellulose membrane, blocking was done using 5% BSA. Primary and Secondary antibodies are mixed in 5% BSA. We used primary antibodies of EpCAM (Santa Cruz, sc-25308), phosphoAKT (Sigma Aldrich, SAB5600064), Total AKT (Sigma Aldrich, SAB5600066), Rad50 (Abcam, ab8913). Ku80 (Cell Signaling Technology, CST2180), phosphoATM (Cell Signaling Technology, CST D6H9), Total ATM (Cell Signaling Technology, CST2873), CHK2 (Cell Signaling Technology, CSTC13C1), Vimentin (Sigma Aldrich V6630), Twist (Santa Cruz, sc-15393) and Tubulin (Abcam, ab7291). Primary antibody of EpCAM has dilution of 1:200, while rests of the antibodies have dilution of 1:1000. We used anti-mouse HRP secondary antibody (ab6728) and anti-rabbit secondary antibody (Thermo Scientific 35512) at 1:10000 dilution.