Clx infrared imaging system

For Western blotting analysis, the indicated amounts of cell extracts were resolved in 12% SDS-PAGE and transferred onto nitrocellulose membranes (Sigma-Aldrich, St. Louis, MO, USA). Normalization was performed by using a monoclonal anti-tubulin antibody (T0198, Sigma-Aldrich, St. Louis, MO, USA), a polyclonal anti-actin (A2066, Sigma-Aldrich, St. Louis, MO, USA) or a monoclonal anti-FLAG (F1804, Sigma-Aldrich, St. Louis, MO, USA). All the other antibodies used were from Novus Biologicals (Abingdon, UK). Blot images were acquired and analyzed by using an Odissey CLx Infrared Imaging system (LI-COR GmbH, Bad Homburg, Germany).

Whole-cell lysates were extracted from HUVECs with sample buffer and boiled at 95 °C for 10 min. Samples were separated by SDS-PAGE, and then transferred to nitrocellulose membrane (Pall, New York, USA). The nitrocellulose membrane was incubated with blocking buffer at room temperature for 1 hour. After discarding the blocking buffer, the blots were incubated with primary antibodies at 4 °C overnight. The primary antibodies used were listed in Supplementary Table 2. 1 × Tris buffered saline with 0.1% Tween-20 (TBST) was used to wash the membrane three times for 10 minutes each time. Then, membranes were incubated with IRDye^® 680RD Goat anti-Mouse IgG (H + L) or IRDye^® 800CW Goat anti-Rabbit IgG (H + L) (1:10,000 dilution, LI-COR, Lincoln, Nebraska, USA) at room temperature for 1 hour. Finally, the Li-COR CLx infrared imaging system was used to visualize the blots.

Day 1 adult worms were washed twice in PBS. Half the worm pellet was resuspended in RIPA buffers with protease inhibitors (Roche, Basel, Switzerland), lysed via sonication, and then used to determine protein concentration with a BCA assay (Pierce, Waltham, MA, USA). The other half of the sample was lysed via sonication in 2x Laemmli sample buffer (BioRad, Hercules, CA, USA) containing 5% beta-mercaptoethanol. From this lysate, 20 ug of each sample was loaded and separated with a 10% tris-glycine gel (BioRad). The separated proteins were transferred to a 0.2 μm nitrocellulose membrane (Invitrogen), then incubated in primary antibodies (phospho-Drosophila p70 S6 Kinase (Thr398), 1:500, Cell Signaling #9209, and beta-actin, 1:1000, MP Biomedicals #8691002) in TBS overnight. The membrane was incubated for 1 h in secondary antibodies (IRDye 800CW Goat anti-rabbit (LI-COR), 1:20,000 and IRDye 680RD Goat anti-mouse, 1:20,000 (LI-COR)). LiCor Odyssey CLx infrared imaging system was used to image the blot and the Odyssey Image Studio software (version 5.2, Lincoln, NE, USA) was used to quantify band intensity.

HUVECs were treated with vehicle (DMSO) or RES (1 μM, 2.5 μM, 5 μM and 10 μM) for 12 h, followed by adenovirus (MOI = 1) infection for another 24 h to overexpress PHACTR1. The treated cells were washed three times with ice-cold PBS buffer to remove the cell culture medium. Whole cell lysate was extracted from HUVECs with 1× sample buffer and boiled at 95 °C for 10 min. Samples were separated by SDS-PAGE and then transferred to nitrocellulose membranes (Pall, New York, NY, USA) and then incubated with blocking buffer (Li-COR, Lincoln, NE, USA) for 1 h at room temperature. After blocking, primary antibody was added and incubated overnight at 4 °C. The primary antibodies used are listed in Supplementary Table S2. The cells were washed 3 times using 1× Tris buffer saline (TBS) containing 0.1% Tween-20 for 10 min each. The membrane was then incubated for 1 h at room temperature with IRDye 680RD goat anti-murine IgG (H+L) or IRDye 800CW goat anti-rabbit IgG (H+L) (1:10,000 dilution, Li-COR, Lincoln, NE, USA). Finally, the blots were visualized using the Li-COR CLx infrared imaging system.