Mini protean tgx gel

Proteins from snap frozen colon samples were combined with Laemmli sample buffer (Bio-Rad Laboratories, Hercules, CA) and 2-Mercaptoethanol (Sigma-Aldrich) and denatured before loading onto a 4–20% Mini-PROTEAN TGX gel (Bio-Rad), and transferred to a polyvinylidene difluoride (PVDF) membrane (Sigma-Aldrich) for immunostaining outlined in the supplement.

MV4:11 cells were treated for 1, 2, 3 or 6 days with 2 μM C6nc and C6, or 0.1% DMSO only. Four million cells were washed in PBS then lysed for 10 minutes on ice in 200 μl Kischkel buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, Protease inhibitor cocktail, 1 μM PMSF). Whole cell extracts were sonicated for 15 s then clarified by centrifugation. Laemmli Sample buffer was added and samples were boiled for 10 minutes before running on a 4%–20% mini-PROTEAN TGX gel (BioRad) and transferring to PVDF membrane. Membranes were blocked in 5% milk in TBST for 20 minutes then probed with appropriate antibodies.

Total protein from cultured neutrophils was extracted using the RIPA buffer (BP-115, Boston BioProdcuts, Ashland, MA) with protease inhibitor and phosphatase inhibitor. Protein concentrations were determined using Pierce BCA assay (PI23223, Fisher Scientific). Equal amount of proteins (20 μg per lane) was separated on a 4-20% Mini-PROTEAN TGX Gel (456-1096, Bio-Rad Laboratories, Hercules, CA). Separated proteins were transferred to PVDF membranes using the Transfer Turbo Blot system (Bio-Rad Laboratories) and the Trans-Blot Turbo RTA transfer kit (170-4272, Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk for 1 hr at room temperature. After blocking, the membranes were incubated overnight at 4 °C with antibodies against IL6 (1:1000, 12912), phosphor-c-Jun N-terminal kinase (p-JNK, 1:1000, 9255), total (t)-JNK (1:1000, 9252,), p-p38 (1:1000, 9211), t-p38 (1:1000, 9212), phosphor-extracellular-signal-regulated kinase (p-ERK1/2, 1:1000, 9101), t-ERK1/2 (1:1000, 9107), and β-actin (1:4000, 4970), all from Cell Signaling Technology. Proteins on the membranes were visualized by chemiluminescence detection kit (Super signal west femto maximum sensitivity substrate, Fisher Scientific). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyze signal abundance.

Protein lysate was run on a 4–20% Mini-Protean TGX gel (BioRad) and transferred onto a nitrocellulose membrane (BioRad). Membranes were incubated overnight at 4 °C with purified anti-MyD88 antibody (ProSci) followed by peroxidase-conjugated donkey anti-rabbit (Jackson ImmunoReseach) and Precision Protein StrepTactin-HRP Conjugate (BioRad). SuperSignal West Pico Chemiluminescent Substrate was used for imaging (Thermo Scientific). Membranes were stripped and incubated overnight at 4 °C with purified anti-β-actin (Invitrogen) followed by peroxidase-conjugated donkey anti-mouse (Jackson ImmunoResearch) and Precision Protein StrepTactin-HRP Conjugate (BioRad).