Bm chemiluminescence blotting substrate kit

Primary fibroblasts were cultured from a skin biopsy sample from Pt1, Pt2, or a control subject in Dulbecco modified Eagle medium supplemented with 10% FBS, 2 mmol/L glutamine, 10 mmol/L HEPES, and 40 μg/mL gentamicin (Life Technologies, Courtaboeuf, France). Fibroblasts were transfected with either pMax-GFP (Lonza, Basel, Switzerland) vector or TrueORF gold vector coding for Myc-DDK-tagged ORF of human AIRE transcript variant AIRE-1 (OriGene Technologies, Rockville, Md) by using the jetPEI reagent (Polyplus Transfection, Illkirch, France). After 24 hours, fibroblasts were lysed in NP-40 lysis buffer (20 mmol/L Tris/HCl (pH 7.4], 150 mmol/L NaCl, 2 mmol/L EDTA, and 1% NP-40 (Sigma-Aldrich]) containing protease inhibitors for 30 minutes at 48C. Supernatants were collected after 10 minutes of centrifugation at 16,000g and 48C, and protein content was quantified with the mBCA quantification kit (Thermo Fisher Scientific Biosciences, Villebon sur Yvette, France). Protein extracts (50 μg) were analyzed by using Western blotting with anti-AIRE antibody (Abnova, Taipei, Taiwan), anti-rabbit IgG antibody conjugated to horseradish peroxidase (1:10,000, Sigma-Aldrich), and a BM Chemiluminescence Blotting Substrate Kit (Roche).

The immunoblotting method for detecting anti-CaSR autoantibodies is described elsewhere.^{E6 (link)} Briefly, 20-mg samples of the Escherichia coli–expressed CaSR extracellular domain (amino acid residues 1-603; SWISS-PROT no. P41180) were separated by means of SDS-PAGE and transferred to nitrocellulose membranes. Membranes were used in standard immunoblotting experiments with patient or control sera (1:100 dilution), anti-human IgG antibody conjugated to horseradish peroxidase (1:2000 dilution, Sigma-Aldrich), and a BM Chemiluminescence Blotting Substrate Kit (Roche). In addition, anti-CaSR autoantibodies were detected in some patients’ serum samples by using a CaSR immunoprecipitation assay, as detailed previously.^{E7 (link)}