Vegfa sc 152

The FDA-approved drug library and allopurinol were purchased from Selleck Chemicals. allopurinol was dissolved with dimethyl sulfoxide (DMSO) for cell experiments. For animal study, allopurinol was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na, purchased from Selleck Chemicals). Cisplatin was purchased from HANSON Pharma and dissolved in 0.9% sodium chloride.
The following antibodies were used in this study: The TMTC3 (sc-398137) and VEGFA (sc-152) antibodies were purchased from Santa Cruz. The β-actin (3700 S) and STAT3 (8768s) antibodies were purchased from Cell Signaling Technology. The HIF-1α (20960-1-AP), IMPDH2 (67663-1-Ig) antibodies were purchased from Proteintech. The p-STAT3 (bs-1658R) antibody was purchased from Bioss. The Flag (F3165) and β-tubulin (T5201) antibodies were purchased from Sigma. The p-JAK2 (ab32101) antibody was purchased from Abcam. The JAK2 (A19629) antibody was purchased from ABclonal Technology.
The siRNAs of IMPDH2 were synthesized by GenePharma (Suzhou, China). The siRNA sequences for IMPDH2 and primers for qPCR were listed in Supplementary Table 1.

Subconfluent cultures were fixed using 4% paraformaldehyde at room temperature (RT) for 10 min, followed by permeabilization with 0.01% Triton ×100 for 4 min. Cells were stained with primary antibodies—YKL-40 (sc-393590), VEGF-A (sc-152), and VEGF-B (sc-1876) from Santa Cruz Biotech, Olig 2 (ab42453), MAP2 (ab11267), ANG1 (ab8451), and ANG2 (ab8452) from Abcam, GFAP (mab 360) from Millipore, and nestin (N5413-R) from Sigma, for 2 h at RT. Subsequently, cells were stained with Alexa Fluor-conjugated secondary antibodies (Invitrogen). For Western blotting, cell monolayers were washed thrice with 1× PBS and harvested using trypsin. Cell pellets were lysed in MPER containing 1× protease inhibitor cocktail (Thermo Scientific). A total of 40 µg of cell lysate was loaded in each experiment, and electrophoresed samples were transferred onto PVDF membrane (Pall Life Science). Membranes were probed using respective primary antibodies (CD44, HPA005787, dilution 1:1,000, Sigma; α-tubulin, T9026, dilution 1:5,000, Sigma; and VEGF-A, VEGF-B, ANG1, and ANG2 at 1:1000 dilution) overnight. Membranes were washed thrice in 1× PBST and probed with secondary antibody (antimouse HRP, 616520, dilution 1:5,000; antigoat HRP, 611620, dilution 1:1,000; and antirabbit HRP, 656120, dilution 1:1,000; Invitrogen) for 2 h at RT. Blots were developed using ECL substrate (Thermo Scientific).