Bca protein assay kit

Metabolic labelling assays were performed as previously described [5 (link)] with the following modifications. Cells were serum starved in DMEM without methionine and cysteine (ThermoFisher Scientific, Cat. 21013024) for 45 min, and pulse-labelled in 100 μCi/mL EasyTag™ Express Protein Labeling Mix, [35S] (Perkin Elmer, Cat. NEG772) for 15 min (WT CFTR) or 1 hour (ΔF508-CFTR). Cells were rinsed with cold PBS containing 1mM MgCl₂ and 0.1mM CaCl₂ and chased in DMEM supplemented with 10% FBS, 2 mM glutamine, 2 mM methionine and 2 mM cysteine. Cells were harvested at 0, 1, 2, or 3 h in 500 μL RIPA buffer. Soluble material was separated by centrifugation at 20,000 x g for 5 min at 4°C. Protein concentrations were normalized using the BCA Protein Assay Kit, and pulse-labelled CFTR was isolated by immunoprecipitation with a mixture of CFTR monoclonal antibodies (Millipore, MAB3480 (1.5 μg) and MAB3484 (1.5 μg) per sample) and 50 μL protein G agarose beads (Millipore, Cat: 16–266) for 2.5 hours at 4°C. Beads were washed 3 times in RIPA buffer and proteins were eluted in Laemmli loading buffer. CFTR bands were separated by 6% SDS-PAGE and radioactive bands were detected using a Typhoon scanner (GE Healthcare), then quantified using ImageJ 1.46r software.

DPC12 Cells were planted into 6-well plates at 4 × 10⁵ cells per well and treated with HE (50 and 100 µg/mL) and NGF (50 ng/mL) for 24 and 48 h. Cells were harvested and lysed with RIPA buffer (Sigma-Aldrich) containing 1% protease inhibitor cocktail (Sigma-Aldrich) and 2% PMSF (phenylmethanesulfonyl fluoride) (Sigma-Aldrich). After protein concentration detection by BCA Protein Assay Kit (Millipore, Billerica, MA, USA), 30 μg of proteins were separated using a 10% SDS-PAGE gel and transferred electrophoretically onto PVDF membranes. After blocking with 5% bull serum albumin (BSA) for 4 h, the transferred membranes were blotted with primary antibodies (β-tubulin III and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) (Abcam, Cambridge, MA, USA) at 4 °C overnight at a dilution of 1:1000, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:2000 (Santa Cruz, CA, USA). Chemiluminescence was detected using ECL detection kits (Millipore) and imaging system (Biospectrun 600). The intensity of the bands was quantified by scanning densitometry using software Image J (National Institutes of Health, Bethesda, MD, USA).

The heart tissues or neonatal mouse primary cardiomyocytes (NMPCs) were lysed with RIPA buffer (P0013B, Beyotime, Shanghai, China) for total protein extraction. The protein concentration was determined using the BCA protein assay kit (71285, Millipore, Bedford, MA, USA) according to the manufacturer’s protocol. 35 μg protein was subjected to SDS-PAGE for electrophoresis, transferred to 0.45 μm PVDF membranes (IPVH00010, Millipore, Bedford, MA, USA), and immunoblotted with antibodies. The bands were quantified using the ImageJ software program (v.1.45, National Institutes of Health, Bethesda, MD, USA). The antibodies are listed in Supplementary Table 1.