Pa5 103744

The total protein was extracted from tissues or cells with radioimmunoprecipitation assay lysis buffer (R0010, Solarbio), with the concentration determined by BCA Kit (20201ES76, YEASEN Biotech Co., Ltd., Shanghai). After separation by polyacrylamide gel electrophoresis, the protein was transferred to the PVDF membrane by wet transfer method. The membrane was sealed with 5% BSA at room temperature for 1 h, probed with the primary antibodies to CMTM7 (#PA5-103744, 1:1000, Thermo Fisher), EGFR (ab52894, 1:1000, Abcam), p-EGFR (ab40815, 1:1000, Abcam), AKT (ab8805, 1:500, Abcam), p-AKT (ab8933, 1:500, Abcam), VEGF (ab32152, 1:1000), CD63 (ab134045, 1:1000, Abcam), Hsp70 (ab181606, 1:1000, Abcam), TSG101 (ab125011, 1:2000, Abcam), and Calnexin (ab133615, 1:1000, Abcam) at 4 °C overnight. The next day, the membrane was re-probed with HRP labeled goat anti-rabbit IgG (ab205718, 1:10,000, Abcam) for 1 h at room temperature, developed by VILBER FUSION FX5 (VILBER LOURMAT, France). Image J 1.48u software (National Institutes of Health) was used for protein quantitative analysis, and the gray value of each protein was compared with the gray value of internal reference GAPDH.

Immunohistochemical staining was used to determine the expression of CMTM7, CD34, CD31, and Ki-67 in the tissues. Cancer tissues were fixed in 4% paraformaldehyde phosphate buffer for 12 h, dewaxed with xylene and hydrated with gradient alcohol (anhydrous ethanol, 95% ethanol and 75% ethanol for 3 min each). The tissues were heated in 0.01 M citrate buffer solution for 15–20 min, and cooled to room temperature, followed by washing with PBS. Thereafter, the tissues were probed with 30 μL primary antibodies against CMTM7 (PA5-103744, 1:200, Thermo Fisher), CD34 primary antibody (ab81289, 1:200, Abcam, Cambridge, UK), CD31 primary antibody (ab134168, 1:100, Abcam) and Ki-67 primary antibody (ab15580, 1:200,Abcam), and then re-probed with 30 μL of secondary antibody goat anti-rabbit IgG (ab6721, 1:2000, Abcam) for 1 h at room temperature. After PBS washing, streptavidin-peroxidase was added dropwise to the tissues and placed at 37 °C for 30 min, followed by DAB development for 5–10 min. Following tap water washing for 10 min, the tissues were counterstaining with hematoxylin for 2 min, differentiated with hydrochloric acid alcohol, dehydrated, cleared and mounted before microscopic observation.