RHBDL4 cleaves SREBP-1c independently of the SCAP-Insig system. A) Schematic diagram of SREBP-1c cleavage by PUFA-sensitive serine protease on the ER membrane. B–F) Indicated proteins were transiently transfected into HEK293 cells (B–E) or CHO-7 and SRD-13A (SCAP−/−) cells (F). After 24 h, cytosolic extracts (CE) and NE were immunoblotted with the indicated antibodies. SREBP-1c cleavage by rhomboid protease family (B), SREBP-1c cleavage by RHBDL4 mutant S144A (C), SREBP-1c cleavage after coexpression of RHBDL4 and Insig1 (D), SREBP-1c mutant 3 M cleavage by RHBDL4 (E), and SREBP-1c cleavage by RHBDL4 in SCAP null cells (F) are shown. HSV-SREBPs were immunoblotted with HSV antibodies. (-), mock; R1, RHBDL1; R2, RHBDL2; R3, RHBDL3; R4, RHBDL4; WT, wild type; SA, the catalytically inactive mutant of RHBDL4 S144A; 3 M, three known cleavage sites for CPP32, S2P, and S1P mutant of SREBP-1c; P, precursor; N, nuclear. G) An in vitro cleavage assay was performed using recombinant HSV-SREBP-1c-HA and RHBDL4-V5 purified from a cell-free protein synthesis system using wheat germ. Indicated proteins were analyzed by immunoblotting. Arrowheads show precursor (P) and nuclear (N) SREBP-1c. H) Eight-week-old male C57BL/6J mice were injected with GFP- or RHBDL4-expressing adenovirus via the tail vein. Six days after injection, the mice (n = 3–4 per group) were subjected to fasting and refeeding. Immunoblot analysis of indicated proteins in whole cell lysates (WCL) and NE from pooled mouse livers was performed.