Ab181602

Total proteins of ischemic brain tissue in each group were extracted by RIPA buffer containing EDTA-free protease inhibitor and phosphate inhibitor (Rocher, Switzerland). After measuring the protein concentration, 30 μg of each sample was subjected to electrophoresis on 15% SDS-PAGE gel at 200 V. The protein was transferred into the PVDF membrane at 100 V in Bio-Rad TransBlot apparatus. 5% skimmed milk diluted by TBST solution was used to block PVDF membranes containing proteins for 2.5 hours at room temperature, and then, the primary antibody, rabbit anti-GADPH (1 : 1000, Abcam, ab181602), NLRP3 (1 : 1000, Abcam, ab181602), caspase 1 (1 : 1000, Abcam, ab181602), and NF-κB (1 : 1000, Abcam, ab181602), were used to incubate PVDF membranes at 4°C overnight. And then, the second antibody HRP-conjugated goat anti-rabbit (1 : 5000; EarthOx, USA) was used to incubate membranes for 2.5 hours after washing three times per 5 mins with TBST solution. Finally, after incubating with enhanced chemiluminescence, the membranes were exposed in ChemiDoc Touch Imaging System. ImageJ software was applied to the quantification analysis of protein bands. Each experiment was performed three times [16 (link)].

The tumour tissue homogenate was treated with 1 ml of lysate (RIPA Lysis Buffer; Applygen Technology Co., Ltd, Beijing, China) and the lysate was transferred to a 1.5‐ml centrifuge tube, followed by centrifugation at 2000 g at 4°C for 5 min. The supernatant was taken for SDS‐PAGE separation, and the separated gel was electroblotted on polyvinylidene fluoride membranes (EMD Millipore Corp., Billerica, MA, USA). After that, the membranes were blocked with dried skimmed milk and probed with the corresponding primary antibodies against YAP (1:10,000; ab181602), TAZ (1:500; ab84927) and GAPDH (1:10,000, ab181602; all from Abcam, Cambridge, UK) and then with the secondary antibody (1:3000; ab205718; Abcam). ImageJ software (v.1.8.0; US National Institutes of Health, Bethesda, MD, USA) was used to perform quantitative optical density analysis on the immunoblot images.

Western blotting was performed using the Simple Western Jess system (ProteinSimple, USA), a combination of capillary electrophoresis and immunodetection techniques, following the manufacturer’s protocols (Beekman et al. 2018 (link)). The left hippocampal samples were collected and were frozen at − 80℃ at 6 h and 7 d after microwave exposure. The hippocampus was homogenized and lysed in a mixed liquid containing radio-immunoprecipitation assay (RIPA) lysis buffer and 1% protease inhibitor after all the samples were collected. The whole process was carried out at a low temperature (0–4℃). A fully automated western blotting was carried out on an array of protein samples after the protein concentration was determined with a bicinchoninic acid (BCA) protein assay kit. Proteins were detected with the following primary antibodies: PSD-95 (ab18258, Abcam), CaMKII (ab134041, Abcam), CREB (ab32515, Abcam), (ab181602, Abcam), GluN1 (MAB363, Millipore Sigma), GluN2A (MAB5216, Millipore Sigma) and GluN2B (ab93610, Abcam). Chemiluminescent signals were captured by a charge-coupled device (CCD) camera, and the resulting images were analysed by Compass software (ProteinSimple, USA) and expressed as peak intensities. For quantification, the areas under the protein peaks were normalized to GAPDH (ab181602, Abcam) and target protein loading controls.