Hb egf

Primary antibodies against ADAM8, EGFR, CCL2, and Akt (panAkt and pAkt S473) were obtained from Proteintech; p-EGFR, ERK1/2 (panERK and pERK1/2 Tyr 202/204) from Cell Signaling Technology (Danvers, MA, USA); HB-EGF from ABclonal technology (Woburn, MA, USA). Iba-1, GFAP, and CD206 were purchased from Abcam (Cambridge, UK). Secondary antibodies were listed in the Supplementary Data Table S1 together with primary antibodies. Temozolomide was purchased from Selleckchem (Houston, TX, USA). As an EGFR inhibitor, we used Erlotinib (Selleckchem).

Total protein was extracted with RIPA buffer, and 20–50 μg samples were loaded after measuring their concentration using a BCA kit and separated by 6%, 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride membrane blocked with 5% fat-free milk for 2 h at room temperature and incubated in primary antibodies against ADAM8 (1:1000, Proteintech), HB-EGF (1:1000, Abclonal Technology), EGFR (1:1000, Proteintech), p-EGFR (1:1000, CST, Beverly, MA, USA), ERK (1:1000, CST), p-ERK (1:1000, CST), AKT (1:1000, Proteintech), p-AKT (1:1000, Proteintech), CCL2 (1:1000, Proteintech), and GAPDH (1:2000, Enogene, New York, NY, USA) at 4 °C overnight. Membranes were washed and incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. An enhanced chemiluminescence system (NCM Biotech, Suzhou, China) was used to detect the protein bands.

Immunofluorescent staining was performed in GBM xenografts and human GBM tissues. Primary antibodies included IBA-1(1:500, Abcam), GFAP (1:200, Abcam), p-EGFR (1:200, Cell Signaling Technology), p-ERK (1:200, Cell Signaling Technology), HB-EGF (1:100, Abclonal Technology), CCL2 (1:200, Proteintech, Rosemont, IL, USA), Briefly, tumor sections were deparaffinized, rehydrated, and antigen-retrieved by standard procedures. Then, samples were blocked with a PBS solution containing 1% BSA plus 0.3% Triton X-100 for 2 h at room temperature and then incubated with indicated primary antibody overnight at 4 °C followed by the fluorescence-conjugated second antibody (1:200) at room temperature for 2 h. After being counterstained with DAPI for 5 min, sections were mounted on glass and subjected to microscopy. The extent of positive cells was measured by ImageJ, which was defined as the ratio of positive cells relative to the total cells in five randomly selected viewing fields. The images were acquired with a fluorescent microscope (Olympus (Tokyo, Japan), CKX53).