Anti ldlr antibody

Cells were lysed using an ice‐cold buffer containing 50 mM Tris–HCl, pH 7.5, 125 mM NaCl, 1% Nonidet P‐40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate, 1 mg/ml complete EDTA-free protease-inhibitor cocktail (Roche), and 0.25 mg/ml Pefabloc, 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride hydrochloride (AEBSF; Roche). Cells were rotated at 4 °C for an hour and centrifuged at 12,000 g during 15 minutes to remove insoluble material. Proteins were fractionated by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative immunoblotting. Following antibodies were added: rabbit polyclonal anti-LDLr antibody (1:500) (Progen Biotechnik GmbH, Heidelberg, Germany), anti-GAPDH antibody (1:1000) (Nordic Biosite, Täby, Sweden) and horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Little Chalfont, UK). The primary antibodies were incubated overnight at 4 °C while the secondary antibody incubation was performed at room temperature for an hour. Signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA) in a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). NIH ImageJ software (http://rsbweb.nih.gov/ij/) was used for band intensity quantification, levels of protein of interest were corrected to GAPDH loading control band intensities. Original blot can be found in Supplementary Fig. S1.

Cell lysates were prepared, using an ice-cold buffer containing 50 mM Tris–HCl, pH 7.5, 125 mM NaCl, 1% Nonidet P-40, 5.3 mM NaF, 1.5 mM NaP, 1 mM orthovanadate, 1 mg/ml protease inhibitor cocktail (Roche), and 0.25 mg/ml Pefabloc, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF; Roche). Cells were sonicated for 10 pulses at 10 kHz on ice, rotated at 4°C for an hour and centrifuged at 12,000g during 15 minutes to remove insoluble material. Proteins were fractionated by electrophoresis on non-reducing 8.5% SDS-PAGE for semi-quantitative immunoblotting. The following antibodies were added: rabbit polyclonal anti-LDLr antibody (1:500) (Progen Biotechnik GmbH, Heidelberg, Germany), anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000) (Nordic Biosite, Täby, Sweden) and horseradish peroxidase-conjugated anti-rabbit antibody (GE Healthcare, Little Chalfont, UK). The primary antibodies were incubated overnight at 4°C while the secondary antibody incubation was performed at room temperature for an hour. Signals were developed using SuperSignal West Dura Extended Substrate (Pierce Biotechnology, Rockford, IL, USA) in a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA). NIH ImageJ software (http://rsbweb.nih.gov/ij/) was used for band intensity quantification, levels of protein were corrected to GAPDH loading control band intensities.