Ab83579

The mouse alveolar macrophage cell line MH-S, and human lung fibroblast MRC-5 cells were obtained from ATCC and cultured in regular DMEM supplemented with 10% FBS (Sigma). For overexpression studies easy-to-transfect human lung osteosarcoma (U2OS) and human embryonic kidney (HEK293) cells were also obtained from ATCC and cultured in DMEM medium with 10% FBS. Human lung adenocarcinoma (NCI-H441 and A549) cells were cultured in RPMI-1640 medium supplemented with 10% FBS. Rabbit polyclonal TTP antibodies were purchased from Abcam Inc. (ab36558 and ab83579). Mouse monoclonal anti-DDK antibody was purchased from Origene (Rockville, MD), and antibodies against β-TrCP1 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Control (Cat. No. D-001810) and β-TrCP1 (Cat. No. D-003463) siRNA were purchased from Thermo Fisher Scientific (Lafayette, CO). P38 kinase inhibitor SB203580 (Cat. V1161) was purchased from Promega Corp. (Madison, WI).

The level of protein in brain tissue homogenate was estimated and was separated by SDS‐PAGE. Then proteins were transferred to polyvinylidene membranes and incubated with primary antibodies for TTP (ab83579; Abcam, Table 1) and PP2A (ab16448; Abcam, Table 1) for 12 hr. The membranes were incubated with HRP‐IgG (goat anti‐rabbit, A0545‐1ML; Sigma‐Aldrich) for 1 hr. TTP and PP2A were assessed by enhanced chemiluminescence (ECL; Zhuang, Ye, & Huang, 2017).

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).