P rps6 s240 244

Western blot analysis was performed as described in our previous studies [28 (link),29 (link)]. Primary antibodies used in the study were as follows: p-AMPK (Thr172) (1:500, cat. # Y408289, ABM, Richmond, BC, Canada), t-AMPK (1:1000, cat. # 2523, Cell Signaling Technology, Danvers, MA, USA), p-ACC (S79) (1:1000, cat. # 2535, Cell Signaling Technology, USA), t-ACC (1:1000, cat. # 3662, Cell Signaling Technology, USA), Caspase-3 (1:1000, cat. # 9661, Cell Signaling Technology, USA), p-rpS6 (S240/244) (1:2000, cat. # 5364 Cell Signaling Technology, Danvers, MA, USA), rpS6 (1:2000, cat. # 2217, Cell Signaling Technology, Danvers, MA, USA), Bax (1:2000, cat. # ab32503 Abcam, Cambridge, UK) and tubulin (1:3000, cat. # CSB-MA000185 Cusabio Biotech, Wuhan City, China). Horseradish peroxidase-conjugated antibodies to rabbit immunoglobulins (1:60000, cat. # 111-035-003, Jackson Immuno Research, Cambridge, UK) were used as secondary antibodies. Following image capture of phosphorylated proteins, membranes were stripped of the phospho-specific antibodies using RestoreTM Western Blot Stripping Buffer (cat. # 21059, Thermo Fisher Scientific, Waltham, MA, USA). The membranes were then re-probed with primary antibodies for each respective total protein. A total protein staining (Ponceau S) and/or tubulin protein expression were used for normalization of Western blots.

The β-actin (1:1000, sc-130656) and BCKDK (E-12) (1:1000, sc-374425) antibodies were purchased from Santa Cruz Technology, Inc. (Santa Cruz, CA, USA). Flag antibody (1:1000, #F1804, #F7425) was purchased from Sigma-Aldrich (St. Louis, MO, USA). His Mouse monoclonal antibody (1:1000, 4E6) was obtained from Pregene, Inc (Beijing, China). All the following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): p-BCKDHA (S293) (1:500, #40368), BCKDHA (1:500, #90198), MEK1/2 (1:1000, #8727), p-MEK1/2 (S221) (1:1000, #2338), p-ERK1/2 (T202/Y204) (1:2000, #4370), ERK1/2 (1:2000, #4695), p-RPS6 (S235/236) (1:1000, #2211), p-RPS6 (S240/244) (1:1000, #2215), RPS6(1:1000, #2217), E-cadherin (1:500, #3195), N-cadherin (1:500, #13116), and Vimentin (1:500, #5741). p-BCKDK (Y246) antibody was prepared by Abiocode, Inc (shanghai, China). Rabbit (1:3000, #E030120) and mouse (1:3000, #E030110) antibodies were obtained from EarthOx Life Sciences (San Francisco, CA, USA). We purchased BT2 (Cat No. ZC-26488) from ZZBIO. CO.LTD (Shang Hai, China). Dasatinib (Cat No. S1021) was purchased from Selleckchem, Inc (Houston, TX, USA).