Pierce bca colorimetric assay

Cells were seeded in 24-well plates with at least three biological replicates per condition, and then treated with drugs and/or transfected with siRNA. Cells were then incubated for 1 hour with 0.05 μCi iodine-125 (¹²⁵I) (Hartmann Analytic; Folkestone, UK) at a final concentration of 10^-7 moles/l NaI. Following incubation, cells were washed twice in HBSS (Sigma-Aldrich) to remove unincorporated ¹²⁵I and lysed in 100 μl 2% SDS. Radioiodide uptake was determined using the Berthold Gamma Counter (Berthold) to measure gamma radiation (counts per minute). Protein concentrations were determined using the Pierce™ BCA colorimetric assay (ThermoFisher Scientific). ¹²⁵I uptake relative to protein concentration was calculated in picomoles of ¹²⁵I/ μg protein, according to the following equation: $\text{Picomoles of }125\text{I/ }\mathrm{\mu }\text{g protein }=\frac{5000\ast \left(\frac{Counts}{TotalProtein}\right)}{12000}$
In control experiments, cells were treated with 100 μM sodium perchlorate for 1 hour at 37°C and 5% CO₂ prior to the assay to inhibit NIS-mediated radioiodide uptake.

Total cell extracts were prepared using RIPA buffer with protease inhibitors (Roche) with additional PMSF (1 mM), sodium orthovanadate (1 mM) and sodium fluoride (50 mM) added just before use. Protein concentration was quantified using the Pierce BCA colorimetric assay (ThermoFisher) and quantified against BSA standards with a Multiskan plate imager at 550 nM. Extracted proteins were resolved on 4-15% TGX gels (Bio-Rad) and transferred to PVDF membranes using iBlot dry transfer system (Invitrogen). Immunoblots were developed with the help of indicated primary antibodies and corresponding HRP tagged secondary antibodies). Blots were scanned using the ThermoFisher myECL imager.