Ripa lytic buffer

HMEC-1 cells were lysed with RIPA lytic buffer (Beyotime Biotechnology, China). The same number of protein samples were separated by 10% SDS-PAGE and transferred onto an NC membrane (Millipore, USA). After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with one of the following primary antibodies: β-actin (bsm-33036M, bioss, 1:1,000), HIF-1α (ab51608, Abcam, 1:1,000), α-SMA (ab5694, Abcam, 1:500), CD31 (ab28364, Abcam, 1:500), VEGF-a (ab32152, Abcam, 1:500), VE-cadherin (YT4869, Immunoway, 1:500), and fibronectin (YM3137, Immunoway, 1:500). The membranes were washed with PBS and incubated for one hour with the secondary antibody. Finally, the electrochemiluminescence (ECL) reagent was used to visualize them (Millipore, USA). ImageJ was used to capture the protein images.

Cell samples were lysed with cold RIPA lytic buffer (#P0013B, Beyotime, China), and the extracted proteins were quantified using Pierce BCA Protein Assay Kit (#P0009, Beyotime, China). After electrophoresis was performed on a sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE), the blots were transferred to a polyvinylidene difluoride membrane (#IPVH00010, Millipore, America). The membranes were incubated with diluted primary antibodies (survivin, #10,508–1-AP, 1:1000, Proteintech; PTPRT, #PA5-18,304, 1:500, Invitrogen) and GAPDH (#60,004–1-Ig, 1:16,000, Proteintech) at 4 °C overnight, and then the second antibody was added and incubated at room temperature for 30 min. ECL reagent (#RM00020, ABclonal, China) was added to the membranes to visualize the immunoreactive protein bands. Notably, the marker (Shanghai Yase Biomedical Technology Co., LTD., WJ102) was not able to be developed. Therefore, the position of the marker on the Kodak film after exposure was determined by comparing the color bands on the acetate fiber film using SDS-PAGE horizontal electrophoresis (Supplementary Fig. 1– 4).