Ab124024

Proximity ligation was performed using the Rabbit PLUS and Mouse MINUS Duolink In Situ PLA kits (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, KYSE180 cells (5 × 10⁴) were plated on coverslips and treated with nicotine or vehicle for 24 h. The cells were then washed twice with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 15 min at room temperature (RT). Subsequently, cells were washed with TBS (25 mM Tris, 100 mM NaCl, pH 7.4), incubated for 10 min in 50 mM NH4Cl, TBS, washed with TBS, permeabilized for 15 min in 0.1% Triton X-100 in TBS, washed with TBST (0.05% Tween 20 in TBS), and then blocked for 2 h with 0.5% milk powder in TBST in a humidified chamber at RT and incubated overnight at 4 °C with anti-OTUD3 (Sigma-Aldrich, HPA028544) and anti-ZFP36 (Abcam, ab124024) antibodies. After washing with TBST, proximity ligation was performed using the PLA kits (Sigma-Aldrich). Cells were further counterstained with 4,6-diamidino-2-phenylindole (Sigma-Aldrich) to visualize the nuclei. The images were obtained by using laser scanning confocal microscopy (LSM880, Carl Zeiss MicroImaging, Oberkochen, Germany).

Frozen spinal cord tissue sections stored in liquid nitrogen were taken out, and proteins were extracted with the addition of 1 ml RIPA lysis buffer (#89901, Thermo Scientific, USA) containing 2% protease inhibitor and 1% PMSF followed by completely homogenisation using an ultrasound homogeniser. Tissue homogenisations were then centrifuged at 12,000 rpm for 10 min, and the supernatants were collected and stored in -80°C for later use. Protein concentrations were quantified using BCA assay. Concentrated protein samples were then loaded into SDS-PAGE gels for electrophoresis and transferred onto 0.45 μm PVDF membranes. Membranes were then incubated with diluted primary antibodies against MK2 (ab131531, Abcam, UK), p-MK2 (ab131504, Abcam, UK), TTP (ab124024, Abcam, UK), NLRP3 (ab263899, Abcam, UK), pro-caspase-1 (ab179515, Abcam, UK), pro-IL-1β (ab205924, Abcam, UK), iNOS (ab15323, Abcam, UK), arg-1 (ab272887, Abcam, UK), and GAPDH (ab8245, Abcam, UK) as loading internal control at 4°C for 12 h. On the next day, membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature after rinse with TBST buffer solution for 3 changes. ECL chemo-reagents were then applied onto the membranes, and membranes were captured using an imaging system. Grey values of protein bands were detected and analysed using the ImageJ software.