SREBP Processing Regulation by Caspase-2

To test SREBP processing, HEK 293 (ATCC® CRL-1573) cells were plated at a density of 0.5 × 10⁵ per well (6-well plates). The next day, 1 μg of each cDNAs were transfected using lipofectamine 3000 (Thermo Fisher Scientific, MA) according to the manufacturer’s instruction. cDNAs used were empty vector (EV), Casp2-HA, PIDD-Flag, RAIDD-His, Myc-S1P, and V5-SREBP2. After 4 h, cells were incubated in DMEM/F12 medium supplemented with indicated Casp2 inhibitors (10 μM) for 16 h. Whole-cell lysates were prepared in lysate buffer (150 mM Tris-HCl, pH 7.4, 10% sodium-deoxycholate, 100 mM NaCl, 100 mM EDTA, 100 mM PMSF, 200 mM NaF, 100 mM Na₃VO₄, and a mixture of protease inhibitors). Membrane fraction and nuclear extracts were prepared as described previously [12 (link)]. Equal quantities of proteins were subjected to immunoblot (WB) analysis with anti-Flag (#F7425 Sigma-Aldrich), anti-HA (#1867431, Roche), anti-Casp2 (#ALX-804-356, 11B4, Enzo Life Sciences), anti-V5 monoclonal antibody (#13202, Cell Signaling Technology), anti-Casp3 (#9661, #9662, #9664, Cell Signaling Technologies), and anti-HSP90 (#sc-101494, Santa Cruz Technologies).