Journal: eLife

Article Title: SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion

doi: 10.7554/eLife.00444

Figure Lengend Snippet: ( A ) Non-reduced plasma protein samples from four wild type and four Sec24a gt/gt mice were separated on SDS-PAGE and stained with coomassie brilliant blue. The first lane contains size markers. Asterisks indicate transferrin and albumin. An ∼25kD protein (p25) is under-represented in the plasma of Sec24a gt/gt mice. Lower panel, quantification of the intensity for the band labeled ‘p25' in the upper panel; error bars represent SEM. Asterisk, p<0.01 by Student's t-test. ( B ) Identification of p25 as APO-A1 by mass spectrometry. Peptide sequences detected in HPLC-ESI-MS/MS analysis; all six peptides exhibit 100% match with mouse APO-A1 sequence. ( C ) Non-reduced plasma protein samples from four wild type or four SEC24A-deficient mice were analyzed by immunoblotting with antibodies to albumin or APO-A1. ( D ) Pooled plasma samples from seven wild type and eight Sec24a gt/gt mice were fractionated by FPLC and cholesterol in each fraction quantified with a colorimetric assay; total cholesterol for the fractions containing HDL, LDL, and VLDL/Chylomicrons are indicated in the table at the bottom. ( E ) Total plasma cholesterol in male wild type control (n = 10 for 8 weeks of age, n = 4 for 6 months of age) and Sec24a gt/gt (n = 5 for 8 weeks of age, n = 6 for 6 months of age). Error bars represent SEM. Asterisk, p<0.001 by Student's t-test. ( F ) Total plasma cholesterol in female wild type control (n = 7 for 8 weeks of age, n = 4 for 6 months of age) and Sec24a gt/gt (n = 5 for 8 weeks of age, n = 5 for 6 months of age). Error bars represent SEM. Asterisk, p<0.001 by Student's t-test. ( G ) Total plasma cholesterol in wild type control (n = 7 for male, n = 5 for female) and Sec24a gt2/gt2 (n = 4 for male, n = 4 for female). Error bars represent SEM. *p<0.0001 by Student's t-test. DOI: http://dx.doi.org/10.7554/eLife.00444.007

Article Snippet: Rabbit anti-APO-A1 antibody and rabbit anti-LMAN1 antibodies were purchased from Stressgen.

Techniques: Clinical Proteomics, SDS Page, Staining, Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing, Western Blot, Colorimetric Assay, Control