B15002

After 30 h of infection, cells were lysed using the RIPA lysis buffer (R0020, Solarbio, China) containing protease and phosphatase inhibitors (B14002 and B15002, Bimake, Shanghai, China); all procedures were performed on ice. Protein G magnetic beads (S1430, NEB China group, Beijing, China) were pre-washed with 30 μL lysis buffer. Cell lysates were incubated with the antibody against phosphorylated Smad1/5/8 for 1 h at 4°C. About 15 μL of pre-washed protein G magnetic beads were then added to the lysates and incubated for 1 h at 4°C. The target complexes were collected with a magnetic stand and washed with lysis buffer. Next, 30 μL lysis buffer was used to elute the proteins from beads. Finally, the proteins were analyzed by western blot assay. Antibody against p-CREB were used for detection.

The cell or tissue samples were lysed using RIPA Buffer (P0013B, Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors (78425, Thermo Fisher Scientific, USA; B15001, B15002, Bimake, USA) and protein extraction was performed according to the manufacturer's instructions. Concentration of the protein was determined using the BCA protein assay kit (23225, Thermo Fisher Scientific, USA). Denatured protein diluted to an equal concentration was separated by SDS-PAGE. Subsequently, the protein was transferred to a PVDF membrane at a constant current of 300 mA. After blocked in 5% skim milk for 1 h, the membrane was incubated overnight with the primary antibody: rabbit anti-GFAP antibody (1:1000; 16825-1-AP, Proteintech, USA), mouse anti-HIF-1α antibody (1:200; SC-13515, Santa Cruz, USA), rabbit anti-IBA1 antibody (1:1000; ab178846, Abcam, USA), mouse anti-TH antibody (1:1000; 22941, Immunostar, USA), mouse anti-β-actin antibody (1:1000; SC-47778, Santa Cruz, USA). The next day, after washing, the membrane was incubated with the corresponding secondary antibody for 1 h, and the band of interest was detected using the Odyssey infrared imaging system (Li-Cor, USA). The relative level of protein was quantified using ImageJ software (NIH, Bethesda, MD).

Cells were lysed in EBC buffer (50 mM Tris pH 7.5, 120 mM NaCl, 0.5% NP-40) supplemented with protease inhibitors (A32953, Thermo Fisher) and phosphatase Inhibitors (B15002, Bimake). The protein concentrations of lysates were measured using the Beckman Coulter DU-800 spectrophotometer and the Bio-Rad protein assay reagent. Same amounts of whole cell lysates were resolved by SDS-PAGE and immunoblotted with indicated antibodies. For immunoprecipitation, cell lysates containing 1 mg of total proteins were incubated with anti-Flag agarose (A2220, Sigma) or anti-HA Agarose (A2095, Sigma) for 4 hours at 4 °C. Precipitants were washed three times with EBC buffer and resolved by SDS-PAGE followed by immunoblot analysis with indicated antibodies.