Bolt 12 bis tris gel

Cell lysates were prepared as outlined previously [1 (link)]. Enteroids were resuspended in RIPA buffer with cOmplete protease inhibitors (Roche), sonicated, then centrifuged at 16,000xg for 20 min at 4°C. Total protein was estimated (660nm Protein Assay; Pierce) and 10 μg of whole cell lysate prepared according to manufacturer’s instructions in 1X Bolt LDS Sample Buffer with 1X Bolt Reducing Agent (Life Technologies) and heated at 70ºC for 10 min. Proteins were separated by Bolt 12% Bis-Tris Gel (Life Technologies), transferred to PVDF membrane (Life Technologies), followed by immunoblotting with mouse monoclonal anti-caspase-11 (p20 Flamy-1;1:1000; AdipoGen), mouse monoclonal anti-caspase-1 (p20 Casper-1;1:2000; AdipoGen), or mouse monoclonal anti-β-actin (G043; 1:2000; Applied Biological Materials), then with horse α-mouse IgG:HRP (7076; 1:2000; Cell Signaling Technologies).

Colonoids were resuspended in RIPA buffer with protease inhibitors and phosphatase inhibitor, sonicated, then centrifuged at 16,000xg for 20 min at 4 °C. Total protein was estimated and 10 μg of whole cell lysate prepared according to manufacturer’s instructions in 1X Bolt LDS Sample Buffer with 1X Bolt Reducing Agent (Life Technologies) and heated at 70 °C for 10 min. Proteins were separated by Bolt 12% Bis–Tris Gel (Life Technologies), transferred to PVDF membrane (Life Technologies), followed by immunoblotting with rabbit monoclonal phospho-p38 (1:2000; Cell Signaling Technologies #9211), total p38 (1:2000; Cell Signaling Technologies #9212), phospho-NFκB (1:2000; Cell Signaling Technologies #3033), total NFκB (1:2000; Cell Signaling Technologies #8242) or mouse monoclonal anti-β-actin (1:2000; ABM # G043), then with horse α-rabbit IgG:HRP (1:2000; Cell Signaling Technologies #7076) or horse α-mouse IgG:HRP (1:2000; Cell Signaling Technologies #7076). Western blot images were taken using a ChemiDoc imaging system (Bio-Rad) and densitometry analysis of the obtained images were done using ImageJ 1.45S software (Wayne Rasband, NIH).

DKK3 or decorin (4 μM) were incubated with 1 µM of HTRA1^FL or 1 µM of HTRA1^PD/PDZ in TNC buffer for 16 h at 37 °C in the presence or absence of CKPs (5 μM). Biglycan (4 μM) was incubated with 0.5 µM of HTRA1^FL or HTRA1^PD/PDZ in TNC buffer for 4 h at 37 °C in the presence or absence of CKPs (2.5 μM). Reduced samples were electrophoresed on Bolt 12% Bis-Tris gels (Thermo Fisher Scientific) in 1x MES SDS Running buffer (Thermo Fisher Scientific) at 200 V for 25 min. Gels were stained in SimplyBlue SafeStain (Thermo Fisher Scientific) and imaged on a Bio-Rad Gel Doc EZ Imager.