P ser555 ulk

Cells were treated with leflunomide for 72 h, then cells were collected, after that proteins were extracted by RIPA Lysis Buffer which contained 1% Phenyl methane sulfonyl fluoride (PMSF), stockpiled at −80°C. The proteins were separated by SDS-PAGE, subsequently, transfered onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with 5% BSA for 2 h, and incubated with a primary antibody against human Tubulin (1:1000, Beyotime), LC3B (1:1000, Cell Signaling), CDK2 (1:1000, Cell Signaling), CyclinA2 (1:1000, Cell Signaling), DHODH (1:1000, Cell Signaling), BCL-2 (1:1000, Cell Signaling), Caspase-9 (1:1000, Cell Signaling), Caspase-3 (1:1000, Cell Signaling), pT172-AMPK (1:800, Cell Signaling), AMPK (1:800, Cell Signaling), pSer555-Ulk (1:1000, Cell Signaling), Ulk (1:1000, Cell Signaling), pThr 183/186-JNK (1:2000, Abcam), JNK (1:1000, Abcam), BCL-2 (1:1000, Cell Signaling) and Beclin1 (1:1000, Cell Signaling) at 4°C overnight. Then incubated with homologous secondary antibodies HRP-labeled goat anti-rabbit IgG (H+L) (1:2000, Beyotime) or goat anti-mouse IgG (H+L) (1:2000, Beyotime) for 2 h. Finally, the signal was captured by the ECL reagent (Beyotime) and visualized by Western blotting detection instruments (Clinx Science).