Western Blot Analysis of FIGF, AKT, and LYVE-1

Cells were harvested in the presence of a protease inhibitor cocktail (Sigma-Aldrich) in RIPA lysis buffer (Beyotime, Jiangsu, China). Equal amounts of proteins from the cells were resolved on SDS-PAGE and then transferred onto PVDF membranes as previously described (6 (link)). The membranes were separately probed with rabbit anti-FIGF (1:1,000, PAB4879; Abnova, Atlanta, GA, USA), rabbit anti-AKT1 (1:1,000, ab32505), rabbit anti-pAKT1 (s473) (1:1,000, ab66138; both from Abcam, Cambridge MA, USA), rabbit anti-mouse LYVE-1 (1:1,000, ab36993; AngioΒio, San Diego, CA, USA), rabbit anti-GAPDH (1:500; Sigma-Aldrich) for 1 h. Subsequently, the membranes were washed with TBST, and then incubated with goat anti-rabbit HRP (1:500; Sigma-Aldrich) for 1 h. Bound antibody chemiluminescence was detected using chemiluminescence kits (Thermo Fisher Scientific, Darmstadt, Germany). The optical density was determined using a scanning densitometer and analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).