Bckdha

Cells were lysed with RIPA lysis buffer. Cytoplasmic and nuclear proteins were isolated with a protein extraction kit (KeyGEN BioTECH, China) according to the manufacturer's instructions. The quantity of protein was measured with a BCA protein assay kit (KeyGEN BioTECH). Protein (10–20 μg) was separated with SDS-PAGE and transferred to nitrocellulose membranes. Non-specific binding sites were blocked with 5% nonfat milk for 2 h, and blots were incubated overnight with primary antibodies to BCKDHA (Cell Signaling Technology, USA), p-BCKDHA (Bethyl Laboratories, USA), HMGB1 (Abcam, UK), p65 (Abcam, UK), IκBα (Cell Signaling Technology, USA), TBP (Abcam, UK), Tubulin (Bioworld Technology, USA) or GAPDH (Bioworld Technology, USA). After being rinsed with PBST, the membrane was incubated with secondary antibodies for 1 h at 37 °C. The protein bands were visualized with an enhanced chemiluminescence reagent (Millipore-Sigma, USA) on a Quantity One system (Bio-Rad, USA).

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).