Arterioscler Thromb Vasc Biol Induction of Ubiquitin-Conjugating Enzyme by Aggregated Low Density Lipoprotein in Human Macrophages and Its Implications for Atherosclerosis by Jiro Kikuchi, Yusuke Furukawa, Nobuhiko Kubo, Akihiko Tokura, Nakanobu Hayashi, Mitsuru Nakamura, Michio Matsuda, and Ikunosuke Sakurabayashi Arterioscler Thromb Vasc Biol Volume 20(1):128-134 January 1, 2000 Copyright © American Heart Association, Inc. All rights reserved.

Nucleotide and deduced amino acid sequences of LIG cDNA. Nucleotide and deduced amino acid sequences of LIG cDNA. Nucleotide positions are numbered on the left with the first base of the putative initiator methionine at +1. Asterisk denotes the stop codon. The HIP-2 cDNA sequence is aligned in lowercase, which shows 100% amino acid identity. The region corresponding to S27–15-(4) is underlined. Arrows indicate the alternatively spliced region. The active site cysteine at residue 92 is italicized. Jiro Kikuchi et al. Arterioscler Thromb Vasc Biol. 2000;20:128-134 Copyright © American Heart Association, Inc. All rights reserved.

LIG/HIP-2 mRNA expression in various human tissues. LIG/HIP-2 mRNA expression in various human tissues. Poly(A)+ RNAs (2 μg per lane) from 8 different normal human tissues (MTN blot II, Clontech Laboratories Inc) were hybridized with 32P-labeled full-length fragment of LIG cDNA (top panel). The sources of RNAs are indicated on top, and the positions of RNA size markers are shown on the left. The membrane was rehybridized with β-actin probe to indicate the amounts and integrity of RNA samples (bottom panel). Jiro Kikuchi et al. Arterioscler Thromb Vasc Biol. 2000;20:128-134 Copyright © American Heart Association, Inc. All rights reserved.

LIG/HIP-2 mRNA expression in human hematopoietic cells. LIG/HIP-2 mRNA expression in human hematopoietic cells. Human hematopoietic cells were isolated from the peripheral blood and bone marrow of healthy volunteers and cultured for 48 hours in the absence (none) or presence of appropriate stimulants: phytohemagglutinin (PHA, 2 μg/mL) for T lymphocytes, PMA (10 ng/mL) for monocytes, and granulocyte-macrophage colony stimulating factor (GM-CSF, 20 ng/mL) for granulocytes. Total cellular RNA was subjected to Northern blot analysis for LIG/HIP-2 mRNA expression (top panel). Ethidium bromide-stained 28S and 18S rRNAs are shown as a loading control (bottom panel). Data shown are representative of 3 independent experiments. Jiro Kikuchi et al. Arterioscler Thromb Vasc Biol. 2000;20:128-134 Copyright © American Heart Association, Inc. All rights reserved.

Induction of LIG/HIP-2 mRNA by aggregated LDL in human macrophages. Induction of LIG/HIP-2 mRNA by aggregated LDL in human macrophages. A, Human peripheral blood monocytes were cultured with PMA (10 ng/mL) alone or with PMA (10 ng/mL) and agLDL (0.5 mg/mL) for up to 48 hours. Total cellular RNA was isolated at the indicated time points and subjected to Northern blot analysis for LIG/HIP-2 mRNA expression. B, Monocytes were cultured in the presence of PMA (10 ng/mL) and various amounts of agLDL (0, 0.1, 0.5, and 1.0 mg/mL) for 48 hours. LIG/HIP-2 mRNA expression was examined by Northern blotting. Ethidium bromide–stained 28S and 18S rRNAs are shown as a loading control (bottom of panels A and B). Data shown are representative of 3 independent experiments. Jiro Kikuchi et al. Arterioscler Thromb Vasc Biol. 2000;20:128-134 Copyright © American Heart Association, Inc. All rights reserved.

Enhanced ubiquitination in agLDL-treated macrophages. Enhanced ubiquitination in agLDL-treated macrophages. Peripheral blood monocytes were cultured with PMA (10 ng/mL) and agLDL (0.5 mg/mL) and harvested at the indicated time points. A, Intracellular ubiquitination was detected by Western blotting in the presence of ubiquitin aldehyde and urea to stabilize ubiquitinated proteins. B, Coomassie brilliant blue staining of the gel was shown to indicate the amounts and integrity of protein samples. Data shown are representative of 3 independent experiments. Jiro Kikuchi et al. Arterioscler Thromb Vasc Biol. 2000;20:128-134 Copyright © American Heart Association, Inc. All rights reserved.

Ubiquitin-dependent degradation of p53 in agLDL-treated monocytes and its role in suppression of apoptosis. Ubiquitin-dependent degradation of p53 in agLDL-treated monocytes and its role in suppression of apoptosis. Monocytes were cultured with PMA and agLDL in the absence [LLnL(−)] or presence [LLnL(+)] of 50 μmol/L LLnL for the indicated periods and lysed in EBC buffer containing protease inhibitors and ubiquitin aldehyde. Immunoprecipitation was carried out with anti-p53 monoclonal antibody, and immune complexes were separated on 10% SDS-polyacrylamide gels, followed by Western blotting with anti-ubiquitin antibody (A). The same samples were subjected to p53 immunoblotting (B). DNA was simultaneously isolated from the cells and electrophoresed on 0.8% agarose gels as previously described23 (C). Data shown are representative of 3 independent experiments. Jiro Kikuchi et al. Arterioscler Thromb Vasc Biol. 2000;20:128-134 Copyright © American Heart Association, Inc. All rights reserved.