Biorad western blot imaging system

Cells were washed with cold PBS and proteins extracted with Laemmli's buffer. Samples were run on 10 or 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with StartingBlock buffer (ThermoScientific) and probed with primary antibodies overnight at 4°C: VEGFR3 (R&D systems), phospho-VEGFR3 (Cell Applications), VEGFR2 (Cell Signaling), PECAM-1 (Abcam), VE-cadherin (Santa Cruz), GFP (Invitrogen) and actin (Santa Cruz). DyLight conjugated fluorescent secondary antibodies (680 nm and 800 nm, Thermoscientific) or HRP-conjugated antibodies were used to detect primary antibodies. Bands were detected and quantified with an Odyssey infrared imaging system for DyLight antibodies (Li-Cor) or a BioRad western blot imaging system (Bio Rad).

Cells were lysed in RIPA lysis buffer (Applygen, China). The protein concentration was detected with a bicinchoninic acid (BCA) kit (Tiangen, China). During electrophoresis, 40 ng of protein was added to each well and run on 8% SDS-PAGE. The proteins in the gel were then transferred onto PVDF membranes (Millipore, Germany). The membranes were blocked with 5% nonfat milk and incubated for 2 h at room temperature. An anti-GAPDH (#2218 S, Cell Signaling Technology, 1:1000), anti-KIAA1429 (#51104, CST, 1:1000), anti-RAB27B (AO0173, Boster, 1:1000), or anti-YTHDF1 (#51104, CST, 1:1000) was then added, followed by overnight incubation at 4 °C. After being rinsed three times, the secondary antibody (Anti-Rabbit, 7074 S, CST, 1:5000) was added, followed by incubation for 2 h at room temperature. Finally, blots were detected using an enhanced chemiluminescence kit (Thermo, USA) and analyzed by a Bio-Rad Western Blot Imaging System (Bio-Rad, Berkeley, CA, USA).