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1.
Fig. 4.

Fig. 4. From: Hepatic lipase maturation: a partial proteome of interacting factors.

Association of the lectin chaperones CNX and CRT with HL in untreated and DTT-treated cells. Cells transfected with (+) or without (−) the HL-TAP construct were treated (+) or not treated (−) with DTT, a reducing agent that causes protein unfolding in the ER. After TAP, the resulting isolates were subjected to Western blot analysis using CNX and CRT antibodies.

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.
2.
Fig. 3.

Fig. 3. From: Hepatic lipase maturation: a partial proteome of interacting factors.

Venn diagram comparing ER proteins copurifying with HL-TAP in untreated CHO cells or cells treated with DTT, a reducing agent that causes protein unfolding. Included is BiP, which also copurified with PL-TAP. The shaded areas represent proteins that are the strongest candidates for roles in HL maturation and degradation.

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.
3.
Fig. 1.

Fig. 1. From: Hepatic lipase maturation: a partial proteome of interacting factors.

Sequence of the human HL-TAP construct. The boxed sequences represent the relevant domains, including the V5 epitope tag and the tandem affinity tag. The TAP tag is comprised of a calmodulin binding domain, a TEV cleavage site, and two IgG binding domains.

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.
4.
Fig. 7.

Fig. 7. From: Hepatic lipase maturation: a partial proteome of interacting factors.

A model of protein complexes formed during HL maturation/degradation. HL is depicted in four states of folding: partially or fully translated HL that is unfolded (gray oval); misfolded HL monomer (wavy gray oval); folded HL monomer (gray hammerhead); and assembled HL homodimer (head-to-tail gray hammerhead). Only the HL dimer exhibits enzyme activity, and it is the only form secreted by the cell. All depicted interacting proteins (while circles) were identified in this study with the exceptions of the Sec61 complex, EDEM, and OS-9, which are inferred.

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.
5.
Fig. 5.

Fig. 5. From: Hepatic lipase maturation: a partial proteome of interacting factors.

HL ERAD occurs via the proteasomal pathway. A: CHO cells expressing HL, PL, or H2b were incubated with or without the proteasomal inhibitors MG132 and MG262. The accumulation of degradation products is clearly seen in H2b (arrowhead), a well-established example of a secretory protein that undergoes proteasome-mediated degradation. Degradation products also appear when HL is treated with either inhibitor, in contrast to PL, showing no detectable degradation via the proteasomal pathway. B: The HL degradation product is unglycosylated. HL from CHO cells incubated with or without the proteasomal inhibitor MG132 was subjected to endo H cleavage. The arrow points to the HL degradation product (two bands).

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.
6.
Fig. 2.

Fig. 2. From: Hepatic lipase maturation: a partial proteome of interacting factors.

A: Representative SDS PAGE gel containing silver-stained proteins isolated by TAP using PL or HL as entry points. The control (Ctrl) lane is a TAP of CHO lysates that have not been transfected with any TAP constructs. Shown on the right are regions of the gel containing some examples of proteins copurifying with an HL-TAP construct (see Tables 1, 2 for a comprehensive list). Only BiP, mitochondrial solute carrier proteins (PTP and ANT2), and GAPDH copurified with the PL-TAP construct. B: All proteins copurifying with HL in all experiments have been combined and presented with regard to location or function. The number of proteins identified in a given class is given in parentheses. The shaded areas represent locations and functions consistent with proteins that may directly or indirectly interact with HL during its co and posttranslational maturation and degradation.

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.
7.
Fig. 6.

Fig. 6. From: Hepatic lipase maturation: a partial proteome of interacting factors.

The sequence and glycosylation of the first N-terminal 60 amino acid residues of human HL lacking the signal peptide. Two N-linked glycosylation sites are present at positions 20 and 56. The glycan structure shown is the unprocessed Glc3Man9GlcNAc2 that is transferred by the oligosaccharyl transferase to the growing polypeptide chain. The first two processing events are removal of the outermost glucose residue by glucosidase I (GI), followed by removal of the second glucose residue by GII. The removal of the second glucose residue from the N-linked glycan at Asn56 is greatly facilitated by transactivation of GII by the N-linked glycan at Asn20 (see text). The resulting Glc1Man9GlcNAc2 is a substrate for CNX binding. The removal of the final glucose residue is also catalyzed by GII, and its removal signals the release of HL from CNX. This second cleavage is not affected by transactivation. Notice the close juxtaposition of the first disulfide bond in HL (Cys40—Cys53) with regard to the second N-glycan chain at Asn56.

Mark H. Doolittle, et al. J Lipid Res. 2009 June;50(6):1173-1184.

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