Image quant las 4000 station

Yeast cells harboring pYC2-CT derivatives encoding BteA-GFP fusion proteins were cultivated in SC-Ura-galactose media for 2, 5, and 20 h to induce the BteA expression. Equivalents of OD₆₀₀ = 1 of yeast cell cultures were collected and denatured yeast protein extracts were prepared by NaOH lysis/TCA precipitation method according to Volland et al. 1994 [41 (link)]. Equal amounts of protein extracts were separated by SDS-PAGE electrophoresis (10% gel) followed by the transfer onto a nitrocellulose membrane. Membranes were probed overnight with mouse polyclonal antibodies raised against BteA (dilution 1:10,000, kindly provided by Branislav Vecerek, Institute of Microbiology, Prague, Czech Republic) or mouse monoclonal antibody against yeast phosphoglycerate kinase PGK1 (anti-PGK1, dilution 1:5,000, Abcam, ab113687) as a loading control. The detected proteins were revealed with 1:3,000-diluted horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody (GE Healthcare) using a Pierce ECL chemiluminescence substrate (Thermo Fisher Scientific, USA) and an Image Quant LAS 4000 station (GE Healthcare, USA).

For preparation of yeast protein extracts, yeast cells with pYC2-CT vectors encoding BteA protein derivatives (Table S4) were induced for 20 h by cultivation in SC-galactose media. Equivalents of OD₆₀₀ = 1 of yeast cell cultures were collected, and denatured protein extracts were prepared by NaOH lysis/TCA precipitation method, according to (45 (link)). For preparation of mammalian cell protein extracts, HeLa cells at 40% confluency in a 6-well plate were transiently transfected with pEGFP-N2 vectors encoding BteA protein derivatives (Table S4) using Lipofectamine 2000 reagent (Invitrogen). Eighteen hours after transfection, cells were washed with PBS, lysed with 100 μl of ice-cold lysis buffer containing 0.2% Triton X-100 and complete mini protease inhibitors (EDTA free, Roche) in PBS, and clarified by centrifugation (5 min, 10,000g). Extracts were mixed with SDS-PAGE sample loading buffer, heated for 5 min at 50 °C, and separated on 10% SDS-PAGE gels. After the transfer onto nitrocellulose membrane, the proteins were probed overnight with rabbit anti-GFP antibody (1:2000; clone D5.1, Cell Signaling Technology) and revealed by horseradish peroxidase–conjugated anti-rabbit IgG secondary antibody (1:3000; GE Healthcare). Blots were developed using a Pierce ECL chemiluminescence substrate (Thermo Fisher Scientific) and an Image Quant LAS 4000 station (GE Healthcare).

Proteins prepared from mouse thyroid tissue collected in RIPA buffer and sonicated were quantified using the BCA protein assay (Thermo Fisher Scientific). Then, 20 μg of total protein was separated on Bis–Tris polyacrylamide gel with a 4–12% gradient (Thermo Fisher Scientific) and transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were incubated with the primary antibodies mouse anti‐Chop (1:1,000, # 2895, Cell Signaling Technology, Danvers, MA, USA) or rabbit anti‐Actin (1:2,000, # A5441, Sigma‐Aldrich) antibodies, followed by horseradish peroxidase‐conjugated goat anti‐mouse or anti‐rabbit antibodies. Binding of secondary antibodies was revealed using the Amersham ECL Prime Detection Reagent Kit (GE Healthcare, Chicago, IL, USA). The protein bands on the membranes were scanned with the ImageQuant LAS 4000 Station (GE Healthcare) and then analysed using ImageJ 1.32s to determine the protein levels, with Actin protein serving as an internal control.