Human vegfa elisa kit

The antibodies used for Western blotting and IP in this study included anti-MDK antibody (sc-46701, Santa Cruz, USA), anti-USP12 antibody (LS-C370534, LSBio, USA), anti-CD31 antibody (77699, CST, USA), anti-Flag antibody (14793, CST, USA), anti-GST antibody (2624, CST, USA), anti-HA antibody (3724, CST, USA), mouse IgG (3420, CST), anti-GST agarose antibody (A8580, Sigma Aldrich) and anti-Flag^® M2 affinity gel (A2220, Sigma Aldrich). MG132 (C2211, Sigma Aldrich) was dissolved in dimethyl sulfoxide. Matrigel matrix (356234, Corning), the human VEGF-A ELISA kit (EKO588-96, Boster) and the human VEGF-C ELISA KIT (EKO539-96, Boster) were also used.

The cell lines HEK293T were got from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines KYSE30 and OE19 were obtained from fenghbio (Hunan, China). Antibodies for ATF5 (ab184923), VEGFA (ab52917), TIMP1 (ab61224), TWIST1 (ab175430), EGF (ab206423), CD31 (ab134168) and POL II (ab264350) were bought from Abcam (Cambridge, MA, USA). Antibodies for PDK1 (13037), PGK1 (68540), CA9 (5649), CBP (7389), P300 (54062), HIF1α (14179), HIF1β (5537) and flag-tag (2368) were procured from Cell Signaling Technology. The human VEGFA ELISA Kit (EK0539) was purchased from BOSTER Biological Technology (Wuhan, China). Identified primers were synthesized by TSINGKE Biological Technology (Beijing, China). For hypoxic conditions, cells were cultured in a sealed hypoxia chamber with a mixture of 1% O2, 5% CO2, and 94% N2 for 2 h.

GIST882 cells, a kind gift of Jonathan Fletcher (Dana-Farber Cancer Institute, Boston, MA), were derived from a GIST patient with a homozygous missense mutation in KIT exon 13 (K642E). GIST-T1 cells obtained from Biowit Technologies (Shenzhen, China) were derived from a GIST patient with an in-frame deletion of 57 nucleotides in KIT exon 11 (V560Y579del). Both cell lines used in this study were cultured at 37 °C with 5% CO₂ in DMEM supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. Antibodies to KDM4D (ab93694) and VEGFA (ab1316) were purchased from Abcam (Cambridge, MA, USA). HIF1a (#36169), H3K36me3 (#4909) and H3K9me3 (#13969) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies to GAPDH (sc-47,724), HIF1β (sc-17,811), Histone3 (sc-517,576) and mouse IgG (sc-69,786) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The human VEGFA ELISA Kit (EK0539) was obtained from Boster Biological Technology (Wuhan, China).