Silencing CALCRL Expression in BON-1 Cells

Endogenous CALCRL-expressing BON-1 cells (DSMZ, Braunschweig, Germany) were seeded onto poly-L-lysine-coated 60-mm dishes and grown to 80% confluence. Cells were either left untreated or treated with chemically synthesised, double-stranded CALCRL siRNA duplexes (Santa Cruz Biotechnology, Dallas, TX, USA) in accordance with the manufacturer’s instructions. A scrambled siRNA was used as the negative control (Santa Cruz Biotechnology). Subsequently, the cells were lysed in detergent buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES, pH 7.4], 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulphate, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL aprotinin, and 10 mg/mL bacitracin). CALCRL enrichment was conducted using wheat germ lectin agarose beads (J-OIL MILLS, Inc., Tokyo, Japan), as previously described [130 (link)]. Subsequently, the protein content of the samples was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions, and the samples (20 µg of protein per lane) were subjected to 7.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene fluoride membranes. Blots were incubated with the rabbit monoclonal anti-CALCRL antibody 8H9L8 (1:500 dilution) overnight at 4 °C, then incubated with peroxidase-conjugated secondary anti-rabbit antibody (1:5000 dilution; Santa Cruz Biotechnology) for 2 h at room temperature and visualised by enhanced chemiluminescence (Amersham, Braunschweig, Germany).
For adsorption controls, the anti-CALCRL antibody was preincubated for 2 h at room temperature with either 10 µg/mL of the immunising peptide (peptide 2) or 10 µg/mL of a control peptide that corresponded to a different region of the receptor (peptide 1; residues 23–40; sequence: ELEESPEDSIQLGVTRNK).