Ecl plus western blot detection reagents

Cell lysates from β-gal assays were normalized based on pre-lysis OD₆₀₀. Lysates were mixed with 6× Laemmli loading dye with PopCulture Reagent (EMD Millipore Corp), boiled for 10 min at 95°C and electrophoresed on 10–20% Tris–glycine gels (Thermo Fisher) in 1× NuPAGE MES Running Buffer (Thermo Fisher). Proteins were transferred to PVDF membranes (BioRad) using a semi-dry transfer system (BioRad Trans-blot Semi-Dry and Turbo Transfer System) according to manufacturer's instructions, and probed with 1:10 000 primary antibody (anti-RpoA-NTD; Neoclone or anti-ProQ; kindly provided by G. Storz) overnight at 4°C and then a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse IgG or anti-rabbit IgG; Cell Signaling, 1:10 000). Note that, throughout the paper, ‘anti-ProQ’ is written out rather than using the standard abbreviation of ‘α-ProQ.’ This is to avoid confusion with the fusion protein we call ‘α-ProQ’ consisting of the NTD of RpoA (α) fused in frame to ProQ. Chemiluminescent signal from bound peroxidase complexes was detected using ECL Plus western blot detection reagents (BioRad) and a c600 imaging system (Azure) according to manufacturer's instructions.

Cardiac cells were lysed by 12,000×g centrifugation for 15 min at 4°C in RIPA buffer (Beyotime Institute of Biotechnology) including a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Subsequently, samples were separated by SDS/PAGE and transferred to PVDF membranes which were incubated overnight at 4°C in 5% BSA with the following antibodies: Calcium Ion Regulation Antibody Sampler Kit (Cell Signaling Technology, Boston, Massachusetts, USA) (1:1,000); GAPDH (Abcam, Cambrige, UK) (1:1,000); and anti-TSHR (Santa Cruz Biotechnology, Dallas, TX, USA) (1:100). Samples were then washed three times with TBST buffer before adding secondary antibody. Using the ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad, Hercules, CA, USA), specific protein bands were visualized using ECL Plus Western blot detection reagents (Bio-Rad). Densitometric analysis of band intensity was completed using Imagelab software (Bio-Rad, Hercules, CA, USA).

Cell lysates from β-gal assays were normalized based on OD600 with LB plus PopCulture Reagent. Lysates were mixed with 6× Laemmli loading dye, boiled for 10 min at 95°C and electrophoresed on 10%–20% Tris–glycine gels (Thermo Fisher) in 1× NuPAGE MES Running Buffer (Thermo Fisher). Proteins were transferred to PVDF membranes (Bio-Rad) using a semidry transfer system (Bio-Rad Trans-blot Semidry and Turbo Transfer System) according to the manufacturer’s instructions. Membranes were probed with 1:10,000 primary antibody anti-ProQ overnight at 4°C and then a horseradish peroxidase (HRP)-conjugated secondary antibody (anti-rabbit IgG; 1:10,000). Chemiluminescent signal was detected using ECL Plus Western blot detection reagents (Bio-Rad) and a c600 imaging system (Azure) according to the manufacturer’s instructions.