Starbright blue 520 and 700

Lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100 and protease inhibitors) was used to lyse the confluent cells after three ice-cold PBS washes. The lysates were centrifuged for 15 min at 13,000× g, and the protein concentrations of the supernatants were measured by a Pierce™ BCA Protein Assay Kit (Thermo Scientific™, Dreieich, Germany). We used 12% TGX gels Stain Free gels (Bio-rad, Feldkirchen, Germany; Cat. 1610181) to separate the proteins before blotting them to nitrocellulose membranes 0.2 µM through a Trans-Blot Turbo Transfer System (Bio-rad, Feldkirchen, Germany). Identifying the proteins was carried out by fluorescence antibodies StarBright Blue 520 and 700 (Bio-rad, Feldkirchen, Germany) using the ChemiDoc MP imaging machine (Bio-Rad, Feldkirchen, Germany). ImageJ software provided by the National Institutes of Health, Bethesda, MD, USA, was used to assess the gray density of Western blots. The signal for the protein of interest is normalized to the total amount of protein loaded into the lane using a stain-free membrane [47 (link)].

After three washes with ice-cold phosphate-buffered saline (PBS), cells were lysed in lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitors) and cell lysates were cleared at 13,000× g for 15 min. Protein concentrations of the supernatants were determined using a Pierce™ BCA Protein Assay Kit (Thermo Scientific™, Dreieich, Germany). Proteins were separated in 7.5% TGX Stain Free gels (Bio-rad, Feldkirchen, Germany, Cat. 1610181) and after transferring to nitrocellulose membrane (Bio-rad, Feldkirchen, Germany, Cat. 1704270) using a Trans-Blot Turbo Transfer System (Bio-rad, Feldkirchen, Germany), proteins were detected with fluorescently labeled antibodies StarBright Blue 520 and 700 (Bio-rad, Feldkirchen, Germany). Imaging of the blots were performed using a ChemiDoc MP (Bio-Rad, Feldkirchen, Germany). Gray density of Western blots was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells were lysed in lysis buffer (50 mM Tris pH 7.4, 5 mM EDTA, 150 mM NaCl, 1 percent Triton X-100, and protease inhibitors) after three washes with ice-cold phosphate-buffered saline (PBS), and cell lysates were cleared at 13,000 g for 15 min at 4 °C. A Pierce^TM BCA Protein Assay Kit (Thermo Scientific^TM, Waltham, MA, USA) was used to determine the protein contents in the supernatants. Proteins were separated on 7.5% TGX Stain Free gels (Bio-rad, Hercules, CA, USA; Cat. 1610181) and transferred to nitrocellulose membranes (Bio-rad, Cat. 1704270) using a Trans-Blot Turbo Transfer System (Bio-rad). Fluorescence antibodies StarBright Blue 520 and 700 were used to identify the proteins (Bio-rad). The blots were imaged with a ChemiDoc MP imaging system (Bio-Rad). ImageJ software was used to determine the gray density of Western blots (National Institutes of Health, Bethesda, MD, USA).