Ab16097

Human monocyte-derived macrophages or differentiated THP-1 cells were cultured on coverslips and stimulated with PMA, LPS, ATP, or LPS plus ATP. Cells were fixed in 4% paraformaldehyde and processed for indirect immunofluorescence staining using the following Abs: mouse anti-human NLRP3 (1:100, ab16097, Abcam, Cambridge, MA, UK), mouse anti-human ASC (1:50, sc-514414, Santa Cruz Biotechnology), rabbit anti-human NCOA6 (1:500, A300-411A, Bethyl Laboratories, Montgomery, TX, USA), mouse anti-vimentin (1:100, ab8987, Abcam), and appropriate secondary Abs, such as donkey anti-mouse IgG Cy3 (1:200, 715-166-151, Jackson laboratory, Bar Harbor, MA, USA) and donkey anti-rabbit IgG Alexa 488 (1:1000, A21206, Invitrogen). The Golgi, ER, and lysosomes were stained using CellLight^TM Reagents BacMam 2.0 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The anti-NCOA6 Abs used in this study recognized the C-terminal end (amino acids 2000 to 2063) of human NCOA6 and thus are specific for only NCOA6α and NCOA6β out of four NCOA6 isoforms (α, β, γ, and δ), as these are the only isoforms that possess the recognized sequence [18 (link)]. Images were taken with a Zeiss LSM 810 microscope and contrast-enhanced using Zeiss ZEN microscope software.

Immunofluorescence staining was performed on 10% v/v formalin fixed, paraffin embedded left colon biopsies. Sections were deparaffinized and subjected to 0.01 M citrate buffer (pH 6.0) antigen retrieval at 121 °C for 4 min in a decloaking chamber. Non-specific binding was blocked by a 1-h incubation in blocking buffer [0.1 mol/L phosphate-buffered saline (PBS)/5% normal goat serum/0.05% Tween-20] at room temperature in the dark. Immunofluorescence staining was performed using specific antibodies against NLRP3 (ab16097, Abcam, Cambridge, MA, USA) and IL-1β (ab9722, Abcam, Cambridge, MA, USA) in the dark, at room temperature for 1 h or alternatively overnight at 4 °C. Sections were then incubated for 1 h in the dark with one or more Alexa Fluor conjugated-secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Section were incubated with 4′,6-diamidino-2-phenylindole Dihydrochloride (DAPI, ThermoFisher Scientific, Waltham, MA, USA) diluted in PBS and mounted with ProLong Gold Antifade (P36930, ThermoFisher Scientific, Waltham, MA, USA). Slides were examined using a FV1200 Laser Scanning Confocal Inverted Microscope (Olympus Australia, Melbourne, Australia).

Total protein was extracted from the cells using whole cell lysis assay kit (KeyGEN, Nanjing, China) according to the manufacturer’s protocol. Equivalent amounts of proteins from each sample were subjected to SDS-PAGE electrophoresis and then transferred to a PVDF membrane (Millipore), blocked in 5% fat-free milk for 1 h at room temperature, incubated with specific primary antibodies overnight at 4 °C with gentle shaking, and followed by detection with enhanced chemiluminescence system (SuperSignal West Femto trial kit, Pierce). Primary antibodies were as follows: E-cadherin (1:300, 3195, CST), Vimentin (1:1000, 5741, CST), NLRP3 (1:1000, ab16097, Abcam), and ACTB (1:5000, AT0001, CMCTAG).