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1.
FIGURE 6.

FIGURE 6. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

Radiation inactivation of EL and EL-EL. The loss of triglyceride lipase activity of EL as a function of radiation dose (megarads) is shown. Three sources of EL were studied as follows: conditioned medium from cells expressing myc-His-tagged wild-type EL that was partially cleaved (◇, wt(cleaved)), conditioned medium from cells expressing profurin to produce EL that was uncleaved (Δ, wt(uncleaved)), and conditioned medium from cells expressing profurin to produce EL-EL that was uncleaved (□, dimer(uncleaved)).

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.
2.
FIGURE 5.

FIGURE 5. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

Western blots of EL and EL-EL under conditions promoting different levels of cleavage. The level of protein expression in HEK293 conditioned media was detected by Western blot using a mouse anti-Myc monoclonal antibody (Ab) and HRP-conjugated secondary antibody. GFP (as negative control), EL, and EL-EL were expressed in HEK293 cells under conditions promoting different levels of cleavage. HEK293 cells stably expressing profurin prevented EL cleavage, and HEK293 cells stably expressing PC5a promote the cleavage of EL.

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.
3.
FIGURE 2.

FIGURE 2. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

EL protein forms immunoprecipitable complex during secretion but not after secretion. HEK293 conditioned media were prepared as follows. Lanes 1 and 4 are from cells expressing GFP; lanes 2 and 5 are mixtures of media from cells separately transfected with either EL-myc-His or EL-FLAG; lanes 3, 6, and 7 are media from cells co-transfected with EL-myc-His and EL-FLAG. Western blots (WB) show supernatants (lanes 1–3) and pellets (lanes 4–6) after immunoprecipitation (IP) with mouse anti-Myc from samples as indicated. Lane 7 shows the pellet after immunoprecipitation with mouse anti-FLAG from conditioned media from HEK293 cells co-transfected with EL-myc-His and EL-FLAG. These Western blots used either an antibody against an EL peptide, FLAG tag, or Myc tag for detection. F indicates the full-length monomer size of EL; N indicates the N-terminal domain of EL, and C indicates the C-terminal domain of EL.

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.
4.
FIGURE 4.

FIGURE 4. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

A, cleavage products of EL monomer and EL dimer. A schematic diagram is shown of all the potential intermediate forms of EL monomer and EL dimer induced by the proteolytic cleavage of EL by proprotein convertases. All the possible forms varying from uncleaved to partially cleaved and fully cleaved in the case of EL monomer and EL dimer are represented. The relative molecular mass of each of these forms in kDa is shown. B, Western blot of EL and EL-EL detected by an antibody against Myc tag. Expression in HEK293 conditioned media of Myc-tagged EL and EL-EL was detected by Western blot using a mouse anti-Myc monoclonal antibody and HRP-conjugated secondary antibody. Schematic diagrams of the different forms of EL detected by the anti-Myc antibody are shown. C, Western blot of EL and EL-EL detected by an antibody against EL. Expression in HEK293 conditioned media of Myc-tagged EL and EL-EL was detected by Western blot using a rabbit anti-human EL polyclonal antibody and HRP-conjugated secondary antibody. Schematic diagrams of the different forms of EL as detected by the anti-EL antibody are shown.

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.
5.
FIGURE 7.

FIGURE 7. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

Molecular model of the EL dimer. Top view of the human endothelial lipase dimer model is shown. The N-terminal “head” domains are shaded in blue; the C-terminal “tail” domains are shaded in red. The activation lid is colored green. The three catalytic residues, serine 169, aspartic acid 193, and histidine 274 are rendered in a space-filling format and located under the activation lid. A collection of exposed hydrophobic residues, Met-351, Leu-382, Pro-383, Ile-386, Val-387, Phe-398, Leu-399, Val-400, Pro-437, Pro-440, and Gly-441, is highlighted in pink on monomer 1. Monomer 2 is rendered so as to reveal the N- and C-terminal domains, hinge, and secondary structure.

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.
6.
FIGURE 1.

FIGURE 1. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

Schematic diagram of EL-EL construct. Shown is a schematic diagram representing EL-EL, the homodimer of EL in a head-to-tail conformation. A short hinge (linker) of 8 amino acid residues (GSIEGRLE) joined the two monomeric subunits of EL using overlap extension PCR. EL-EL contained sequences coding for the signal peptide (SP) of EL, the mature EL protein, the 8 amino acid-peptide linker containing a factor Xa site, and another mature EL protein. Each monomeric subunit of mature EL protein has an N-terminal domain (N) and a C-terminal domain (C). To allow comparison of the levels of expression of the dimeric construct (EL-EL) and wild-type EL, we constructed C-terminal myc-His-tagged proteins.

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.
7.
FIGURE 3.

FIGURE 3. From: Identification of the Active Form of Endothelial Lipase, a Homodimer in a Head-to-Tail Conformation.

A, sucrose gradient centrifugation of wild-type EL. After 5–20% sucrose gradient ultracentrifugation, samples were collected. Each fraction was tested for TG lipase activity (♦) and G6PDH activity (■). The percentage of sucrose was also measured in each fraction collected. B, molecular weight of wild-type EL determined by ultracentrifugation. The molecular standards used in this experiment were cytochrome c (12.5 kDa), ovalbumin (45 kDa), malate dehydrogenase (74 kDa), G6PDH (114 kDa), and catalase (240 kDa). After centrifugation (22 h at 200,000 × g at 4 °C), fractions were collected and assayed for enzyme activity and protein concentration. There is a linear relationship between the molecular weights of standard proteins and the sucrose percentage.

Nathalie Griffon, et al. J Biol Chem. 2009 August 28;284(35):23322-23330.

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