Low Density Lipoprotein Receptor

mAb B4/7 was derived from a mouse immunized with a protein fraction isolated from detergent extracts of MDCK cells using fusion proteins containing the cytoplasmic domain of low density lipoprotein receptor. Binding of GEF-H1 to this fusion protein was not specific. Hybridoma production and subcloning were as described (Hauri et al., 1985 (link)). Five subclones of the originally isolated hybridoma line were isolated and found to recognize all the same protein in immunoblots and to produce the same pattern in immunofluorescence experiments.
All peptides and antipeptide antibodies were produced by Gramsch Laboratories. The following peptides were used to generate rabbit polyclonal antibodies: anti–cGEF-H1 NH₂ terminus, MSRIESLTRARTERC; anti–cGEF-H1 COOH terminus, CDFTRMQDIPEETES; anti–cGEF-H1 alternative domain, CRGHDRLDLSVTIRSVH; anti–ZO-1, YTDQELDETLNDEVC; and anti–claudin-4, PRTDKPYSAKYSAAC. The peptides were conjugated to epoxy-activated Sepharose (Amersham Biosciences), and the antibodies were affinity purified as described (Balda et al., 1996 (link)). For α-tubulin, mAb 1A2 was used (Kreis, 1987 (link)), and in some immunofluorescence experiments ZO-1 was detected with rat monoclonal R40.76 (Anderson et al., 1988 (link)) or with a rabbit polyclonal antibody (Sheth et al., 1997 (link)). α-Catenin was detected with the M12K rabbit polyclonal antibody (Herrenknecht et al., 1991 (link)). GTPases were detected by immunoblotting using the following anti-GTPase antibodies: Rho, rabbit polyclonal antibody sc-179 anti-RhoA (Santa Cruz Biotechnology, Inc.) and Rac1, mouse mAb 102 (BD Transduction Laboratories).

Ligand uptake assays were performed on cells seeded onto 35-mm dishes on the day preceding the experiment. On the day of the experiment, the dishes were placed on ice and were washed briefly with prechilled serum-free medium (SFM; DME and 20 mM Hepes containing 1% BSA). The cells were then incubated on a rocker for 30 min at 4°C in 0.6 ml SFM containing either ¹²⁵I-labeled EGF or ¹²⁵I-labeled transferrin (both obtained from PerkinElmer) at a concentration of 500 nCi/ml. The dishes were washed six times with ice-cold SFM, and the surface counts were collected for the zero time point by incubating the cells for 5 min at 4°C in 0.8 ml ice-cold acid wash (0.2 M acetic acid and 0.5 M NaCl), followed by a brief rinse with another 0.8-ml acid wash. The other dishes were incubated with 1.5 ml prewarmed medium (DME containing 10% FCS) at 37°C for various times. Endocytosis was stopped by placing the cells on ice, collecting the medium, and rinsing the cells with ice-cold SFM. Label associated with the cell surface was then collected by acid wash, as above. Finally, the intracellular fraction was collected by extracting the cells twice with 0.8 ml 1 M NaOH. The radioactivity in the medium, acid wash, and NaOH extract was quantified using a gamma counter (Nuclear Enterprises).
We also attempted to follow the fate of ¹²⁵I-labeled LDL, but found that >50% of the prebound ligand dissociated within the first few minutes of warm-up. Therefore, we made use of HeLaM cells expressing a chimera of the extracellular and transmembrane domain of CD8 fused to the tail domain of the LDL receptor. Human CD8 cDNA in pBlueScript^® was a gift from Gudrun Ihrke (University of Cambridge, Cambridge, UK; Ihrke et al., 2001 (link)). The cytoplasmic tail of the mouse LDL receptor (from the arginine residue at position 813) was amplified by PCR from an EST (Clone ID 2581960; GenBank/EMBL/DDBJ accession no. gi6519196) obtained from the I.M.A.G.E. Consortium, incorporating an AflII site into the 5′ end. The resulting fragment was ligated to the AflII site at the end of the transmembrane domain coding sequence of CD8, and the chimera was cloned into pIRES2Neo (CLONTECH Laboratories, Inc.). The construct was sequenced to confirm that a correct in-frame fusion had been achieved, and was then transfected into HeLaM cells. Stably transfected cells were selected and maintained in the presence of 500 μg/ml G418 (GIBCO BRL).
To monitor uptake of the chimera, the cells were treated as above, but incubated with SFM containing 1:100 diluted anti-CD8 (153–020; Ancell Corp.) instead of with a radiolabeled ligand, and the incubation was for 45 min at 4°C instead of for 30 min. The cells were then washed and incubated for a further 45 min at 4°C with SFM containing ¹²⁵I-labeled protein A (1:1,000 diluted; Amersham Biosciences). The rest of the experiment was performed exactly as above.

All animal experiments were performed in compliance with the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center under animal protocol #010-2016 (Boston, MA, USA). Female and male low-density lipoprotein receptor-deficient (Ldlr^−/−) and sortilin^−/− mice, on a high-fat, high-cholesterol diet (HF/HC), underwent AV wire injury (AVWI) (n = 10–12). Echocardiography was performed to evaluate AV function, while multiphoton and confocal imaging was utilized to quantify AV fibrosis and microcalcification.