Iblot gel transfer stacks pvdf regular

Cells were lysed in RIPA buffer, with protease/phosphatase inhibitors, and sonicated to extract protein. Protein concentration was measured using the BCA reagent (Thermo Fisher Scientific) and protein samples were run on NUPAGE 4%–12% Bis-Tris Midi Gels (Life Technologies, NP0321) along with 10 μL of Chameleon Duo Pre-stained protein ladder (LI-COR, 928-60000). The protein was transferred to a nitrocellulose membrane using the iBlot Transfer machine and iBlot gel transfer Stacks PVDF Regular (Life Technologies, 1340109). The membranes were blocked in Odyssey TBS blocking buffer (LI-COR, 927-50000) containing 0.1% Tween 20 for 1 hour at room temperature (RT). Membranes were incubated overnight at 4°C with the following primary antibodies: pRIPK1 (phospho-S166; 1:1000; Cell Signaling #31122 and #65746), RIPK1 (1:1000; BD Biosciences #610459 and Cell Signaling #3493), pMLKL (phospho-S345; 1:1000; abcam #ab196436 and phospho-S358; 1:1000; Cell Signaling #91689), MLKL (1:1000; Cell Signaling #14993 and #37705), MBP (1:1000; abcam ab62631), caspase-8 (1:1000; Cell Signaling #9746), and β-actin (1:5,000; Sigma-Aldrich #A5441). Membranes were incubated with LI-COR secondary antibodies at a 1:5000 dilution, imaged on the LI-COR Odyssey CLx and quantified with FIJI.