Ab109081

Proteins were extracted from HFLs using RIPA lysis buffer (Beyotime, P0013B) and quantified by the BCA protein assay kit (Beyotime, P0012S). After separation by 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis, the proteins were transferred onto a Hybond-P membrane (Amersham Biosciences). The membrane was blocked with 8% skim milk powder in TBST for 1 h at 25 °C, and then incubated with rabbit anti-human pCRTC1 antibody (Sigma, ABE560, dilution 1:1000), rabbit anti-human pCRTC2 antibody (Abcam, ab109081, dilution 1: 1000), rabbit anti-human CRTC2 antibody (Cell Signaling Technology, 9198, dilution 1: 1000), rabbit anti-human pCREB antibody (Abcam, ab32096, dilution 1: 1000) and GADPH (Cell Signaling Technology, 5174, dilution 1: 2000) at 4 °C for 12 h. After extensive washing with 1% TBST, the membrane was incubated with the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, 7074, dilution 1: 5000) for 1 h at room temperature. Bands were visualized with an enhanced chemiluminescence detection kit (Thermo, WP20005). Quantitative analyses were performed using Image J software, with GADPH as the internal standard [15 ]. Images of full-length western blots presented in supporting information.

The protocol was described in the previous study [13 (link)]. Total protein from AML cells was extracted by Radio-Immunoprecipitation Assay (RIPA) buffer, followed by addition with 1× loading dye and boiling for 10 min. Then, 20 μg protein samples were loaded into 10% dodecyl sulfate,sodium salt-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membrane, and blocked with 5% defatted milk powder for 1 h at room temperature. After that, the membrane was impregnated in primary and secondary antibodies. Lastly, chemiluminescence was conducted using a highly sensitive electrochemiluminescence solution (Thermo Fisher, USA). The specific primary antibodies used in this study are as follows: anti-Cyclin D1 (ab40754, Abcam), anti-Bcl2 (ab32124, Abcam), anti-Bax (ab32503, Abcam), anti-mTORC2 (ab109081, Abcam), anti-ZFP36 (ab83579, Abcam), anti-Ki-67 (ab92742, Abcam).