Anti casp3

Proteins were isolated with a lysis buffer composed of 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton X-100, 0.1% SDS and the Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, Waltman, MA, USA). The BCA protein assay kit (Thermo Fisher Scientific) was used to quantify the protein content. An equal amount of protein from each sample was separated on CriterionTM Precast Gel Tris-HCl (Biorad, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA), as previously described^{55 (link)}. The membranes were blocked for 2 hours in 5% non-fat dry milk PBS with 0.1% Tween 20 (Sigma-Aldrich) at room temperature and incubated overnight at 4 °C with primary antibody. After washing, the membranes were incubated for 1 hour at room temperature with horseradish-peroxidase-conjugated secondary antibody. The following primary antibodies were performed: anti- Collagen IV (1:1000, Abcam), anti-Fibronectin (1:1000, Abcam), anti-Vitronectin (1:1000, Abcam), anti-CASP3 (1:1 000, Cell Signaling Technology), anti-CASP9 (1:500, Cell Signaling Technology), anti-BAX (1:1000 Cell Signaling Technology), anti-RHO (1:1000 Merck Millipore), anti-Vinculin (1:1000 Thermo Fisher Scientific). Vinculin was used as the loading control.

After 48 hrs of doxycycline induction, 10 μg total protein was boiled for 5 mins in Laemmli buffer (125 mM Tris pH6.8, 5% β-Mercaptoethanol, 2% SDS, 10% Glycerol, 0.002% Bromophenol Blue). Samples were loaded per lane onto SDS-PAGE gels alongside 3 μl BluEye Prestained protein ladder (GeneFlow). Gels were transferred to PVDF membrane (GE Healthcare) at 350 mA for 1 hr. Membranes were blocked in TBS-T buffer (1x TBS (Severn Biotech), 0.1% v/v Tween-20 (Sigma)) containing 5% non-fat milk powder for 1 hr at room temperature. Primary antibodies were incubated in TBS-T with 1% milk as follows; M2 anti-FLAG (Sigma), anti-CASP3 and anti-HA tag (both Cell Signaling), were used at 1:1000 dilution, β-Actin (Sigma) at 1:4000 dilution, with all incubated at 4 °C overnight. Secondary HRP-linked antibodies (anti-mouse and anti-rabbit, both Cell Signaling) diluted 1:1000 in TBS-T with 1% milk were applied for 1 hr at room temp. Blots were incubated with ECL reagent (Pierce) and developed on film (Sigma) with X-O-Graph developer system.

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).