SUPPLEMENTAL MATERIAL Supplemental methods Cell culture + ...

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Jan 30, 2013 - ANGPTL4 WT, E40K, CC/AA, and GSGS in pcDNA3.1/V5-His-TOPO were a gift ... sequences of ANGPTL4 were PCR-amplified and cloned into ...
SUPPLEMENTAL MATERIAL Supplemental methods Cell culture +transfections HEK293 cells were cultured using DMEM (Lonza) supplemented with 10% FCS and 1% Penicillin/Streptomycin (P/S). Cells were plated into 6-well plates using P/S-free growth medium and subsequently transfected once cells reached 90% confluency. Transfections were performed using 10 μl Lipofectamine 2000 and 4 μg DNA per well according to the manufacturer’s protocol. 12h posttransfection the culture medium was replaced by DMEM supplemented with 1% P/S. 48h posttransfections cell culture medium was collected and cell lysates prepared using 250 μl 50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS supplemented with 1x EDTA-free complete protease inhibitor (Roche). Cloning ANGPTL4 WT, E40K, CC/AA, and GSGS in pcDNA3.1/V5-His-TOPO were a gift from JC Cohen, PhD, UT Southwestern, Dallas, USA. Full length (FL) (aa 1-406), N-terminal (N-ter) (aa 1-160), Cterminal (C-ter) (aa 165-406), and C-terminal + signal sequence (C-ter +SS) (aa 1-22 + 165-406) sequences of ANGPTL4 were PCR-amplified and cloned into pcDNA3.1/V5-His-A using the following primers containing a KpnI (forward primers) or NotI (reverse primers) restriction site: flANGPTL4_for: ATA GGT ACC ATG AGC GGT GCT CCG, flANGPTL4_rev: ATG GCG GCC GCT GGA GGC TGC CTC TGC, N-ter ANGPTL4_for: ATA GGT ACC ATG AGC GGT GCT CCG, N-ter ANGPTL4_rev: ATA GCG GCC GCT GGC AGG CTT GGC CAC, C-ter ANGPTL4_for: ATC GGT ACC ATG CTG CCC GAG ATG GCC, C-ter ANGPTL4_rev: ATG GCG GCC GCT GGA GGC TGC CTC TGC. C-ter +SS ANGPTL4 insert was generated using a two-step deletion PCR strategy using primers ATG ATA GGT ACC ATG AGC GGT GCT CCG & CTC GGG CAG AGC GCT CAG TAG CAC GGC and CTG AGC GCT CTG CCC GAG ATG GCC CAG & ATA ATG GCG GCC GCT GGA GGC TGC CTC TGC in two separate reactions to amplify the SS and the FLD region and primers ATA GGT ACC ATG AGC GGT GCT CCG & ATG GCG GCC GCT GGA GGC TGC CTC TGC in a subsequent ligation PCR. Western Blot Cell lysate and medium were separated on 10% SDS-PAGE gels and subsequently blotted onto PVDF membrane (Millipore, Amsterdam, Netherlands) overnight. To avoid non-specific binding PVDF membranes were submerged in 5% (m/v) skimmed milk powder (SMP) in PBS-T for 1 hour at room temperature. Anti-V5 (Invitrogen, #R960-25) and anti-mouse HRP were diluted 1:5000 in 5% SMP/ PBS-T and allowed to bind for 90 and 60 minutes at room temperature, respectively. Recombinant proteins Full length recombinant ANGPTL4 (4487-AN) and C-ter ANGPTL4 (3485-AN ) were from R&D systems and dissolved as indicated by the manufacturer. Recombinant N-ter ANGPTL4 (ALX-201373) was purchased from Enzo Life Sciences.

Supplemental results The negative correlation between the change in plasma ANGPTL4 and plasma TG levels prompted us to study which form of ANGPTL4 is measured by the ELISA, as ANGPTL4 is known to exist in at least two truncated forms1. ANGPTL4 levels were assessed in lysates and medium of HEK293 cells transfected with various constructs of human ANGPTL4 (Supplemental figure 1A). Although a cleavage resistant mutant form of ANGPTL4 (GSGS) was less effectively secreted compared to wildtype ANGPTL4, it was effectively detected by the ELISA, indicating that the ELISA detects full length ANGPTL4 (Supplemental figure 1B). Defective oligomerization of ANGPTL4 (CC/AA) or LPL inhibition by ANGPTL4 (E40K) had no impact on the ELISA measurement. The ability of the ELISA to recognize full length ANGPTL4 as well as the C-terminal truncated fragment of ANGPTL4 was confirmed using specific deletion constructs (Supplemental figure 1C). In contrast, the N-terminal truncated fragment of ANGPTL4 did not yield any signal. Similar results were obtained using purified full length and truncated recombinant ANGPTL4 proteins (Supplemental figure 1D). Finally, we checked to what extent the plasma ANGPTL4 levels based on ELISA corresponded with the intensity of the bands obtained by immunoblot using an antibody directed against the N-terminus of ANGPTL4, which detects the N-terminal truncated fragment of ANGPTL4. No correspondence was observed. Taken together, these data indicate that the ELISA measures full length and the C-terminal truncated fragment of ANGPTL4 but not the N-terminal truncated fragment.

Supplemental figures

Supplemental Figure 1: ANGPTL4 ELISA does not detect the N-terminal truncated form of ANGPTL4. (A) Western Blot on lysates and medium of HEK293 cells transfected with expression vectors encoding full-length WT ANGPTL4 and mutants E40K (defective in LPL inhibition), CC/AA (defective oligomerization), and GSGS (defective cleavage) fused to V5 and blotted with anti-V5 antibody. (B) ELISA for ANGPTL4 on the same samples as in A. (C) ELISA for ANGPTL4 was performed on medium and lysate from HEK293 cells transfected with expression vectors encoding full length ANGPTL4, the N-terminal truncated form of ANGPTL4, or the C-terminal truncated form of ANGPTL4 (with or without signal sequence). (D) High concentrations of purified recombinant full length ANGPTL4 and C-terminal ANGPTL4 were analyzed by the ANGPTL4 ELISA (left panel). Low concentrations of purified recombinant C-terminal and N-terminal ANGPTL4 were analyzed by the ANGPTL4 ELISA (right panel).

Supplemental Figure 2: ANGPTL4 ELISA does not detect the N-terminal truncated form of ANGPTL4. (A) Western Blot on lysates of HEK293 cells transfected with expression vectors encoding full length ANGPTL4, the N-terminal truncated form of ANGPTL4, or the C-terminal truncated form of ANGPTL4 (with or without signal sequence) using a rabbit polyclonal antibody directed against the N-terminal epitope CQGTEGSTDLPLAPE of human ANGPTL41. (B) Plasma ANGPTL4 levels of 8 subjects were determined by ELISA. (C) Plasma of the same 8 subjects was subject to immunoblotting using the rabbit polyclonal antibody directed against the N-terminus of ANGPTL4.

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Mandard S, Zandbergen F, Tan NS, Escher P, Patsouris D, Koenig W, Kleemann R, Bakker A, Veenman F, Wahli W, Müller M, Kersten S. (2004) The direct PPAR target FIAF/PGAR/ANGPTL4 is present in blood plasma as a truncated protein that is increased by fenofibrate treatment. J. Biol. Chem. 279:34411-20.