Rabbit anti vegfa

NXT was kindly provided by Xianyang Buchang Pharmaceutical Co. Ltd (Shan’xi, China). Rabbit anti-VEGFA, PCK1, GCK, INSR, PI3K (p110), IRS1 and pi-IRS1 polyclonal antibodies were purchased from Proteintech Group (Chicago, IL). Goat anti-AGE polyclonal antibody was purchased from Novus Biologicals (Littleton, CO). Rabbit anti-AKT, pi-AKT, pi-IRS1, IRS2, PI3K (P85), AMPKα and pi-AMPKα polyclonal antibodies were purchased from Cell Signaling Technology Inc (Danvers, MA). The following antibodies were purchased from Santa Cruz Inc.: rabbit anti-MMP2, fibronectin, G6Pase, FGF21, WT1, TGFβ1, TGFβR2 and Smad2/3 polyclonal antibodies; goat anti-MMP9, collagen type I α2 (COL1A2), collagen type IV α1/3 (COL4A1/3), pi-Smad2/3 and CTGF polyclonal antibodies; and mouse anti-GLUT4 monoclonal antibody. The mouse insulin ELISA assay kit was purchased from ABclonal Inc. (Wuhan, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) except as indicated.

Cells were washed with 1× PBS for 3 minutes, fixed with 4% paraformaldehyde for 20 minutes and washed for three times with 1× PBS for 3 minutes. Then, cells were treated with 0.5% Triton X‐100 for 20 minutes at room temperature (this step was omitted when the antigen is expressed on the cell membrane) and washed with 1× PBS. Blocking was performed by normal goat serum for 30 minutes, followed by addition of primary antibody: rabbit anti‐E‐cadherin (1:400 dilutions, ProteinTech, China), anti‐N‐cadherin (1:300 dilutions, ProteinTech), rabbit anti‐Vimentin (1:200 dilutions, ProteinTech), rabbit anti‐VE‐cadherin (1:200 dilutions, ProteinTech) and rabbit anti‐VEGFA (1:200 dilutions, ProteinTech) to incubate overnight at 4°C, and then washed for three times with PBS. Add 1:200 fluorescent secondary antibody: goat anti‐Rabbit Alexa 488 (1:200 dilutions, ZSGB‐BIO, China), goat anti‐Rabbit Alexa 594 (1:200 dilutions, ZSGB‐BIO) and incubated at 37°C in dark for 2 hours. Add DAPI, incubated in dark for 5 minutes, washed with PBS for three times, and observed under a fluorescence microscope.