Ab131531

Anti-A1AR antibody (ab82477), anti-active caspase-3 (ab2302), Chk pAb to albumin (ab106582), Rb pAb to MAPKAP2 (ab131531), Rb pAb to phosphor MAPKAP2 (ab63378), Ms mAb to Hsp27 (ab2790), Rb pAb to phosphor Hsp27 (ab5594), Rb mAb to NeuN (ab177487), anti-ERK1 (phospho T202) + ERK2 (phospho T185) antibody (ab201015), anti-ERK1 + ERK2 antibody (ab17942), anti-JNK1 (phospho T183) antibody (ab47337), anti-JNK1 antibody (ab110724), anti-GFAP antibody (ab10062), and Ms mAb to NeuN (ab104224) were purchased from abcam. Anti-A2aAR antibody (sc-32261), anti-A2bAR antibody (sc-28996), anti-A3AR antibody (sc-13938), β-actin (sc-47778), and GAPDH (sc-365062) were purchased from Santa Cruz. Rb pAb to P38, phosphor P38 was purchased from Cell Signaling.

Deparaffinised spinal cord tissue sections were rehydrated and applied with 3% H₂O₂ solution and rinsed with PBS buffer. Antigen retrieval was done with applying 0.1 M sodium citrate solution. Then, sections were blocked with goat serum with a 30 min incubation at 37°C. Residual serum was removed by then. Primary antibodies against NeuN (#702022, Invitrogen, USA), iNOS (PA1-036, Invitrogen, USA), arg-1 (PA5-29645, Invitrogen, USA), IBA-1 (ab5076, Abcam, UK), NLRP3 (MA5-32255, Invitrogen, USA), and MK2 (ab131531, Abcam, UK) were applied to the sections followed by the overnight incubation at 4°C. After the removal of diluted primary antibody solutions, sections were rinsed with PBS buffer solution, ant incubated with fluorescent-labelled secondary antibody solution at 37°C for 30 min. DAPI staining solution was performed onto the sections for cell nucleus staining after thorough rinse with PBS buffer. Antifade reagent was applied to the sections after that to block the sections. Sections were then observed with a fluorescent microscope.

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.