Flag antibody

The 293T cells were plated on six-well dishes (5 × 10⁶ cells per well) and transfected with 3 μg DNA plasmid (1.5 μg for each expression vector; if the target expression vector was insufficient, parallel empty plasmid was added). At 48 h post-transfection, the cells were lysed using western and IP lysis buffer (cat.: P0013, Beyotime Biotechnology). The lysate was incubated with primary antibodies (0.5∼1 μg Flag antibody (cat.:66008-2-Ig, Proteintech)) at 4 °C overnight and then incubated with Protein G Sepharose (cat.: 17061802, GE) for an additional 4 h at 4 °C. Analysis was conducted using SDS-PAGE electrophoresis followed by western blotting. The results were visualized using the ECL method (cat.: #C900376 and #C900377, Sangon Biotech (Shanghai) Co. Ltd).

Cells were harvested and lysed using RIPA buffer containing 1% PMSF protease inhibitor and phosphatase inhibitor. Protein concentrations were quantified using the BCA Protein Assay kit (Beyotime Biotechnology, Shanghai, China). A certain amount of protein was separated using a jacase preset gel and the proteins were electrotransferred to a PVDF membrane, blocked with skim milk for 2 h, and then incubated with primary antibodies overnight at 4 °C. The next day, the membranes were washed with a TBST solution 3 times/15 min, followed by incubation with the corresponding secondary antibodies for 2 h at room temperature. The membranes were washed again with TBST, 3 times/15 min, after which exposure was performed with ECL (Beyotime Biotechnology, Shanghai, China). Image J/FIJI was used for quantification.
The antibodies against ESM1 (Cat#ab103590), c-Met (Cat#ab216574), p-c-Met (Cat#ab68141), HIF1α (Cat#ab179483), and GAPDH (Cat# ab8245) were purchased from Abcam (Cambridge, MA, USA). The flag antibody (Cat# 20543-1-AP) and C-Myc antibody (Cat#10828-1-AP) were obtained from Proteintech (Wuhan, China). VEGFA (Cat#50661), MMP9 (Cat#13667T), p-RAF (Cat#9421), RAF (Cat#9422), p-MEK (Cat#9154), MEK (Cat#4694), p-ERK1/2 (Cat#4370), ERK1/2 (Cat#9108), p-P38 (Cat#4511), P38 (Cat#9212), p-JNK (Cat#9255), and JNK (Cat#9252) were bought from Cell Signaling Technology (Danvers, MA, USA).

Compounds were obtained from Enamine with a purity over 90% (Supplementary Table S1). We used LPS (Solarbio, Beijing, China; L8880); CCK8 assay kit (Beyotime, Shanghai, China; C0038); TNF-α and IL-6 ELISA kits (Invitrogen, Carlsbad, CA, United States ); ERK (9102S), P-ERK (9101S), P38 (8690T), P-P38 (4631S), JNK (9252T), P-JNK (4668T), MyD88 (D80F5), GAPDH (2118S), rabbit IgG (7074P2), and mouse IgG (7076P2) antibodies (CST, Shanghai, China); HA antibody (Santa Cruz, CA, United States ; sc-7392); FLAG antibody (Proteintech, Wuhan, China; 20543-1-AP); Ly6G antibody (Servicebio, Wuhan, China; GB11229); F4/80 antibody (Servicebio, Wuhan, China; GB11027); and Prime Script™ RT reagent kit (Takara, Shiga, Japan; RR047A).