Mk 2206 2hcl s1078

The following primary antibodies were used: AKT (rabbit, Proteintech, 10176-2-AP, WB1:1500, IP:1:250, RIP:1:100); FUS (rabbit, Abcam, ab23439, WB1:2000, IP1: 200, RIP1:100); phospho-Akt (Ser473) (rabbit, Cell Signaling, #4060, WB1:1500); phospho-Akt (Thr308) (rabbit, Cell Signaling, #13038, WB1:1500); hexokinase I (rabbit, Cell Signaling, #2024, WB1:1000); hexokinase II (rabbit, Cell Signaling, #2867, WB1:1000); Flag (mouse, Sigma-Aldrich, F1804, IP 1:200); GAPDH (mouse, Sangon, D190090, WB 1:5000); H3 (rabbit, Beyotime, AH433, WB 1:500); and p53 (mouse, Active Motif, 39739, WB 1:1000, RIP 1:150). MK-2206 2HCl (S1078) was purchased from Selleck.

For measuring the effects of UBE2T on invasion capacities, matrix-coated transwell invasion assay was performed as previously described [36 (link)]. Briefly, approximately 1×10⁵ NPC cells were suspended in serum-free medium and added to the top chamber of the 24-well, matrix-coated, transwell chamber system (Corning, Cambridge, MA, USA). Normal cell culture medium was added to the bottom chamber and cells were incubated for 24 hours at 37°C. The membrane was fixed and stained. Cells on the membrane were removed and the number of cells below the membrane were counted (5 random × 200 fields per well). For migration assay, the general transwell chamber was used instead of matrix-coated transwell chamber. Additionally, 2 μM AKT inhibitor (MK-2206 2HCl, S1078, Selleckchem, Houston, TX, USA) was used in the transwell analysis.

Rapamycin (Rapa, R8781), 3-methyladenine (3-MA, M9281), bafilomycin A1 (Baf-A1, B1793), antibodies against ATG12 (WH0009140M1), MAP1LC3B (L7543), and ATG5 (WH0009474M1) were purchased from Sigma-Aldrich (Shanghai, CHINA), and MK-2206 2HCL (S1078) purchased from Selleckchem (Houston, TX, USA). Antibodies against Akt (9272), mTOR (2972), AMPK (2532), phospho-Akt (Ser473) (4060), LAMP1 (9091), phospho-mTOR (Ser2448) (5536), phospho-S6 ribosomal protein (Ser235/236) (2211), ribosomal protein (2217), phospho-c-Met (Y1234/Y1235) (4033), and c-Met (4560) were obtained from Cell Signaling Technology (Beverly, MA, USA), whereas antibodies against β-actin (sc-10731), VacA (sc-25790), CagA (sc-17450), phospho-tyrosine (PY99) (sc-7020) and SQSTM1 (sc-28359), and siRNAs specific for SQSTM1 (human, sc-29679), ATG12 (human, sc-72578), c-Met (human, sc-29397), and ATG5 (human, sc-41445), along with a control siRNA (sc-44230) were obtained from Santa Cruz Biotechnology (CA, USA).