Ab184092

Western blots were run by SDS-PAGE at 225 V for 39 min in Novex 4–20% Tris-glycine Mini Wedge 12-well gels (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membrane (NCM) using iBlot 2 Dry Blotting System (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). NCMs were then blocked using 1× Blocker^TM fluorescent buffer (ThermoFisher Scientific, Waltham, MA, USA) for 1 h. Following blocking, 1:500 dilution of a primary antibody including EDA-Fn (SAB4200784; Millipore Sigma, Burlington, MA, USA), TSP-1 (ab85762; Abcam, Cambridge, MA, USA), and α-SMA (ab5694; Abcam, Cambridge, MA, USA), β-actin (ab184092; Abcam, Cambridge, MA, USA), vinculin (ab207440; Abcam, Cambridge, MA, USA), or vimentin (ab92547; Abcam, Cambridge, MA, USA) was added overnight at 4 °C. The NCMs were washed three times with Tris-Buffer Saline with 1% tween (TBST) for 5 min, followed by a 1 h incubation with the secondary antibody (1:2000); donkey anti-rabbit AlexaFluor 568 (ab175470; Abcam, Cambridge, MA, USA), at room temperature. The NCMs were imaged with the iBright FL 15,000 imaging system (ThermoFisher Scientific, Waltham, MA, USA) and analyzed with the iBright analysis software (ThermoFisher Scientific, Waltham, MA, USA).

U-2OS cells, at a 70-80% confluency, were harvested by scraping from a 6-well plate. Cells were lysed and rotated in RIPA buffer (20 mM Tris-HCl at pH 7.5-7.7, 150 mM NaCl, 1% NP-40, 0.5% DOC, 0.05% SDS) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, 4693159001) for 30 min at 4°C. Samples were analyzed by SDS-PAGE electrophoresis. G3BP1 (Bethyl, A302-033A, 1:1000), G3BP2 (Abcam, ab86135, 1:1000), eIF2α (Cell Signaling Technology, 9722S, 1:1000), p-eIF2α (Abcam, ab32157, 1:1000), GFP (Thermo Scientific , A-11122, 1:1000), Caprin-1 (Proteintech, 15112-1-AP, 1:1000), β-actin (Abcam, ab184092, 1:5000) were detected by western blot. Secondary antibody (LI-COR Biosciences, 926-32211, 1:10000) was detected with a LI-COR Odyssey DLx imaging system.