Las 1000 imager

ECs were treated with AS for 24 h, and Western blot was performed according to a previously described method [25 (link)]. Briefly, ECs were harvested and lysed in protein-extraction solution (Intron Biotechnology, Inc., Kyunggi, Korea) containing protease inhibitors and phosphatase inhibitors for 10 min at 4 °C. The total protein concentration in the supernatants was measured using the Bradford assay [11 (link)]. After heating at 95 °C for 5 min, total protein samples (30 μg) were subjected to 6% to 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) at 100 V for 60 min to 100 min. The membranes were incubated with 5% bovine serum albumin in Tris-buffered saline with 0.05% Tween 20 (TBST) for 30 min at room temperature, and then with diluted primary antibodies (1:200–1:1000) in 5% BSA in TBST overnight at 4 °C. The membranes were washed three times with TBST and incubated with the corresponding secondary antibodies. Protein bands were detected using an enhanced chemiluminescence detection kit (Intron Biotechnology, Inc.) and a LAS-1000 Imager (Fuji Film Corp., Tokyo, Japan). Semi-quantitative analysis of densitometry results was performed using Image J software (National Institutes of Health, Bethesda, MD, USA).

Forty-eight hours after transfection, medium was collected and centrifuged at 13,800× g for 5 min at 4 °C. Cells were washed in phosphate-buffered saline buffer (PBS) and lysed in ice-cold RIPA lysis buffer, supplemented with cOmplete Mini protease inhibitor cocktail tablets. The cells were then incubated 30 min on ice and centrifuged at 13800 × g for 15 min at 4 °C. The lysate (detergent-soluble) fraction was removed and the pellet washed twice with PBS. To all fractions were added LDS sample buffer and NuPAGE reducing agent (Invitrogen), heated to 56 °C for 15 min (5 min of boiling at 96 °C for the pellet fraction), separated by SDS-PAGE (NuPAGE, 4–12% Bis-Tris gels), and transferred to PVDF membranes using XCell IITM Blot Module system (Invitrogen) according to the manufacturer’s manual. Specific antibodies and enhanced chemiluminescence were used to detect and visualize the proteins of interest. Blots were visualized using a LAS-1000 imager and Image Reader LAS-1000 Pro v2.6 software (Fujifilm, Tokyo, Japan), and quantification of protein bands was performed using Multi Gauge v3.0 software (Fujifilm).

HeLa cells were lysed in lysis buffer (50 mM Tris-HCl (pH7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 2 mM EDTA, supplemented with protease inhibitor cocktail (Roche)). Lysates were mixed with 2x sample buffer (62.5 mM Tris-HCl (pH6.8), 2% SDS, 10% Glycerol, 5% 2-mercaptethanol, 0.02% Bromophenol blue), electrophoresed on Laemmli SDS-PAGE gel, and transferred onto PVDF membranes (Immobilon-P, Millipore) using a Bio-Rad Trans-Blot system. Membranes were blocked with 1% ECL prime Blocking Reagent (Cytiva) in TBS (137 mM NaCl, 2.6 mM KCl, 25 mM Tris-HCl (pH7.4)) with 0.1% Triton X-100 for 30 min, followed by incubation with primary antibody in blocking buffer overnight at 4 °C. Horse radish peroxidase-conjugated secondary antibodies were incubated for 60 min at room temperature and developed using ECL prime (Cytiva). Blots were imaged with a LAS 1000 imager (Fujifilm). All Western blots were reproduced at least three times as biological replicates. Source data are provided as a Source Data file.