Fig. 3: Flag-SREBP1c cleavage reporter vector construction and validation in vitro. | Communications Biology

Fig. 3: Flag-SREBP1c cleavage reporter vector construction and validation in vitro.

From: Lipolysis-derived fatty acids are needed for homeostatic control of sterol element-binding protein-1c driven hepatic lipogenesis

Fig. 3: Flag-SREBP1c cleavage reporter vector construction and validation in vitro.

A The pCDNA3.1-Flag-SREBP-1c vector contains a triple Flag-tag in front of the human SREBF1 cDNA (Flag-SREBP-1c) and was transiently transfected into HepG2 cells (Fig. 3A). 48 hours after transfection, HepG2 cells were incubated in lipid depleted medium (5% LPDS) or 5% LPDS plus the addition of 100 μM saturated fatty acids (16:0) or unsaturated fatty acids (18:1, 18:2, 16:1 or 20:4, respectively), or the SREBP-1c suppressor 25 hydroxycholesterol (25-HC), and incubated for 16 h. Non-transfected HepG2 cells were used as Flag-negative control (n.t). 2 h before harvest, the protease inhibitor ALLN (N-acetyl-leucinyl-leucinyl-norleucinal) was added. B Whole cell extracts were subjected to Western Blot (WB). P- and N-SREBP-1c were detected using anti-Flag antibody. Band intensities were measured using ImageJ, NIH. Relative levels of proteolytically cleaved N-SREBP-1c were calculated as the relative fraction of N-SREBP-1c/P-SREBP-1c signal intensities and are presented in the dot plot, n = 6/group.

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