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. Author manuscript; available in PMC: 2019 Sep 20.
Published in final edited form as: Cell. 2018 Sep 13;175(1):133–145.e15. doi: 10.1016/j.cell.2018.08.020

Figure 6: Casp2 cleaves S1P to initiate SREBP processing.

Figure 6:

A) Indicated expression vectors were transfected into HEK293 cells. After 48 hr, cells were incubated with 25 μg/ml N-acetyl-leucine-leucine-norleucinal (ALLN) for 3 hr before harvest. WCL were IB’d with antibodies to SREBP1 and 2. B) Indicated expression vectors were transfected into SCAP-ablated HEK293 cells (293ΔSCAP). After 5 hr, cells were incubated with 1% lipoprotein-deficient serum (LPDS) for 16 hr, followed by ethanol or 50 μM mevalonate + 5 μg/ml cholesterol for 12 hr and 3 hr with ALLN before harvest. WCL, membranes, and NE were IB’d. Red stars: N-terminal Myc epitope-containing S1P polypeptides. C) Intracellular localization of Casp2-HA, S1P-Myc and Flag-SREBP2 in S1P-ablated HEK293 cells (293ΔS1P). Transiently expressed Casp2, S1P, and SREBP2 were visualized by immunofluorescence with HA, Myc, and FLAG antibodies, respectively. Nuclei were counterstained with DAPI. Scale bar: 10 μm. D) Indicated proteins were expressed in HEK293 cells. After 48 hr, WCL and culture supernatants (CS) were IB’d with Myc and S1P antibodies (2 lanes per condition). E) Indicated proteins were expressed in 293ΔSCAP cells. 5 hr after transfection, the cells were incubated with 1% LPDS for 16 hr followed by 3 hr with ALLN. WCL, membranes, and NE were prepared and Casp2 and S1P were IB’d with HA and Myc antibodies. CS were IB’d for S1P. Arrow: cleaved Casp2, red stars: N-terminal S1P fragments that retained the Myc epitope. F) ER and Golgi fractions were isolated by differential centrifugation from livers of LFD-fed 7-week-old MUP-uPA and Casp2−/−/MUP-uPA mice. Proteins in each fraction were de-glycosylated with PNGase F and IB’d as indicated. Star: nonspecific band present in the Golgi fraction of Casp2-null liver, F.L: full-length, C2-Cl.: Casp2-cleaved, A-Cl.: autocleaved. G) BL6 hepatocytes introduced with Adv carrying GFP or uPA. After 24 hr, ER, Golgi and nuclear fractions were isolated by differential centrifugation and IB’d for indicated proteins. F.L: full-length, Cl.: cleaved, L.E: long-exposure, S.E: short-exposure. H) IB of S1P in sera of HFD-fed (12 weeks) mice (n = 3) of indicated genotypes. I) Sera from normal individuals, NAFLD and NASH patients (n = 3 per group) with different liver fibrosis scores were IB’d with S1P antibody.