Pab4879

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).

Cell lysate was prepared as previously described and equal amount of protein was used in western blot analysis (18 (link)). Cells were harvested in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), resolved in SDS-PAGE, transferred to polyvinylidene fluoride (PVDF) membrane and probed with antibodies: mouse anti-Sulf2 (2B4; Novus Biologicals, Littleton, CO, USA), rabbit anti-GAPDH (Sigma, St. Louis, MO, USA); rabbit anti-FIGF (PAB4879; Abnova, Atlanta, GA, USA), rabbit anti-PGF (PAB8004; Abnova), rabbit anti-CD82 (ab109529; Abnova), rabbit anti-NR43A (ab92777; Abnova), anti-mouse HRP (Sigma) and anti-rabbit HRP (Sigma).