Goat anti mouse igg alexa 680

72 h after siRNA transfection, HFFs were lysed using RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, and 50 mM Tris, pH 8) supplemented with 1× protease inhibitors without EDTA (Roche). Cleared lysates collected were combined with an equal volume of 2× reducing sample buffer, heated to 95°C for 5 min, resolved on 4–12% Tris-glycine polyacrylamide gel (Thermo Fisher Scientific), and then transferred to nitrocellulose (0.45-µm pores). Secondary antibodies: goat anti-rabbit immunoglobulin G (IgG) Alexa 680 and goat anti-mouse IgG Alexa 680 (Thermo Fisher Scientific). Western blots were quantified by normalizing the signal intensity of the band of the protein of interest to the corresponding GAPDH signal and determining the change in expression of the siRNA-­mediated protein knockdown relative to the control.

48 h after transfection with siRNA, HT1080s were lysed in RIPA buffer (1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 150 mM NaCl, and 50 mM Tris, pH 8) plus 1× protease inhibitors without EDTA (Roche). Cleared lysates were combined with an equal volume of 2× sample buffer, heated to 95°C for 5 min, resolved by SDS-PAGE on a 4–12% Tris-glycine polyacrylamide gel (Thermo Fisher Scientific), and transferred to nitrocellulose (0.2-µm pores; Thermo Fisher Scientific). Secondary antibodies used for Western blotting were: goat anti–rabbit IgG Alexa 680 and goat anti–mouse IgG Alexa 680 (Thermo Fisher Scientific). Blots were visualized on an Odyssey Scanner (LI-COR Biosciences). Western blots were quantified by normalizing the signal intensity of nesprin 3, vimentin, or lamin A to the corresponding GAPDH signal and determining the change in expression of the siRNA-mediated protein knockdown relative to the control.