5 research outputs found
Identification of IF1 in human serum.
<p>(A) Recombinant IF1 (rIF1) or low-abundance protein-enriched serum or HepG<sub>2</sub> cell lysates were subjected to immunoprecipitation with pre-immune antibodies (PI) or anti-IF1 antibodies (Ab). Analysis was performed by Western blotting using antibodies against IF1. On the left panel, 1 µg rIF1 was loaded as a control of molecular weight. Arrow points at 2 bands immunoprecipitated with specific anti-IF1 antibody. (B) (a) Multiple Reaction Monitoring of EQLAALKK peptide of rIF1 (high panel), blank run before the serum sample (middle panel) and serum sample (low panel). Note the scale difference between rIF1, the serum sample and blank run. Three parent/fragment transitions are monitored for each peptide (represented in three different colors). (b) Manually annotated MS/MS fragmentation spectra of EQLAALKK peptide of human IF1 from serum sample.</p
Correlations between IF1 levels and lipid parameters in serum.
<p>Serum lipid levels were compared from both sides of IF1 median (IF1 median = 0.49 µg/mL). Values are expressed as means and (SD); n = 100 normolipemic male subjects from the study population.</p><p>*Indicates significant difference (p<0.05; # after log transformation).</p
Typical standard curve used for IF1 quantification.
<p>Competitive ELISA was performed using rIF1-coated plates (0.5 µg/mL) and biotinylated anti-IF1 polyclonal antibody pre-incubated with different dilutions of rIF1 (0 to 2.5 µg/mL). The colorimetric signal (OD 450 nm–OD 570 nm) is expressed as a part of the maximum signal obtained without rIF1, B/B0. The standard curve shows that fifty percent displacement of binding was obtained at a concentration of 0.4 µg/mL rIF1.</p
Correlation between IF1 levels and HDL-C (panel A) and TG (panel B).
<p>All individual data of measurements in 100 normolipemic male subjects are represented. Regression coefficients (r) and statistical significance (p) are given.</p
Characterization of polyclonal anti-IF1 antibody.
<p>(A) Sensorgrams are representative of specific interactions (differential response) while nonspecific binding (with no protein immobilized) was deduced. rIF1 was injected at a concentration ranging from 12.5 to 400 nM. Results are expressed as resonance units (RU) as a function of time in seconds. The apparent kinetic constants of the interaction were k<sub>a</sub> = 3.87e<sup>5</sup> M<sup>−1</sup> s<sup>−1</sup>, k<sub>d</sub> = 2.37e<sup>−3</sup> s<sup>−1</sup>, and K<sub>D</sub> = 6.12e<sup>−9</sup> M. (B) HeLa cells were incubated or not with Mitotracker for 30 min at 37°C. Cells were then fixed and processed for immunofluorescence using anti-IF1 antibody. (C) HeLa cells were fixed and processed for immunofluorescence using both anti-IF1 and anti-alpha subunit of ATP synthase (alpha) antibodies. In (B) and (C), the merged images demonstrate perfect overlap between IF1 and both Mitotraker and alpha chain of mitochondrial ATP synthase. (D) HeLa cells were treated with siRNAs against IF1, fixed and then processed for immunofluorescence using anti-IF1 antibody and DAPI for nuclear stain. (B–D) Scale bar indicates 10 µm.</p