Bca kit

Total protein was extracted from both the tissues and cells in strict accordance with the instructions of the efficient radio‐immunoprecipitation assay (RIPA) lysis buffer (R0010, Beijing Solarbio Life Sciences Co Ltd). Next, protein concentration was determined in each sample using a bicinchoninic acid (BCA) kit (20201ES76, Yeasen Biotechnology Co Ltd). Protein transfer onto a polyvinylidene fluoride (PVDF) membrane was performed following isolation by polyacrylamide gel electrophoresis (PAGE) and subsequently sealed with 5% BSA at room temperature for 1 hours. Incubation was subsequently conducted on the membrane with diluted primary rabbit anti‐human antibodies (Abcam Inc) to BTG2 (ab85051, 1:800), vascular endothelial growth factor (VEGF, ab39256, 1:1000), VEGF receptor (VEGFR, ab36844, 1:1000), matrix metalloproteinase‐2 (MMP‐2, ab37150, 1:1000) and MMP‐9 (ab73734, 1:1000) overnight at 4℃. The membrane was then further incubated with HRP‐labelled goat anti‐rabbit IgG diluent (ab205718, 1:20 000, Abcam Inc) at room temperature for 1 hours. ImageJ 1.48 u software (National Institutes of Health) was applied for protein quantitative analysis.

Cells were collected at the specified times and washed twice with PBS. The cell extracts were prepared using lysis buffer and then centrifuged at 13,000 × g for 30 minutes at 4 °C. The protein concentration was quantified using a BCA kit (Yeasen, Shanghai, China). The supernatant was collected, and 5× SDS protein loading buffer was added in proportion, followed by incubation at 100 °C for 10 minutes. Proteins were separated on a 10–12% SDS‒PAGE gel and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA). The membranes were blocked with 5% nonfat milk in Tris‐buffered saline supplemented with 0.1% Tween 20 at room temperature for 2 hours. The primary antibody was diluted with 1× TBST according to the instructions and incubated overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies for another 2 hours at room temperature. Immunoblots were visualized using an enhanced chemiluminescence detection kit (Yeasen, Shanghai, China) with a chemiluminescence imaging analysis system (Tanon, Shanghai, China). Relative integrated density values were calculated using ImageJ software. The following antibodies were used: anti-cGAS, cGAS (E5V3W); rabbit mAb #79978 (CST, Boston, MA); anti-NF-κB, p65 (D14E12); XP® rabbit mAb #8242 (CST, Boston, MA); and GAPDH (Sigma‒Aldrich, Louis, MI).

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.