Ab58705

Immunofluorescence staining of mouse colon sections was performed as previously described (26 (link)). The sections were incubated with primary antibodies against VDR (ab109234, Abcam), NLRP6 (ab58705, Abcam), Caspase-1 (ab138483, Abcam) and ASC (ab175449, Abcam) at 4°C overnight, followed by an incubation with fluorescently labelled secondary antibodies. The sections were examined using a Leica confocal microscope (LEICA TCS SP5).

Briefly, cells were lysed in RIPA buffer supplemented with protease inhibitors (Thermo Scientific), and lysates were sonicated and then centrifuged to remove debris. Equal amounts of total protein in sample buffer were loaded on SDS-polyacrylamide gels and processed for immunoblotting using the following antibodies: NALP3 (Adipogen AG-20B-0014, 1:1000), NALP6 (Abcam ab58705, 1:1000), Nalp7 (Abcam ab126979, 1:2000), Pro-IL-1β (R&D Systems clone 615417, 1:1000), caspase-1 (R&D Systems mAB615, 1:1000), and β-actin (Sigma clone AC-15, 1:10,000). Immunoblots were exposed to SuperSignal West Femto chemiluminescent substrate (Thermo Scientific).

The collected MIECs and mouse colon tissues were extracted using protein lysis buffer (Sigma-Aldrich, USA) and quantified via a bicinchoninic acid assay (Pierce, USA). Protein samples were then electrophoresed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF, EMD Millipore, MA, USA), which was probed with antibodies against VDR (ab109234, Abcam), NLRP6 (ab58705, Abcam), Caspase-1 (ab138483, Abcam) and ASC (ab175449, Abcam) at a dilution of 1:1000. Blots were subsequently detected and visualized using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA) according to protocols provided by the manufacturer. A Bio-Rad scanning system was used to detect immunoreactive protein bands, and GAPDH (ab204276, Abcam) was used as a control.