Nci5079

The brain cortex was harvested 24 h after MCAO/R for western blotting. Brain tissue was homogenized in RIPA buffer (0.1 g/ml) (P0013B, Beyotime, Shanghai, China) at 4°C. The protein concentration was measured using BCA assay (P0010, Beyotime). Equal amounts of protein were loaded in the sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and the following antibodies were employed: anti-fat mass and obesity-associated protein (FTO) (1:1,000, abclonal, Wuhan, China), anti-GAPDH [1:1,000, Cell Signaling Technology (CST), MA, United States] and HRP-conjugated secondary antibody (1:3,000, CST). Protein blots were visualized with a chemiluminescence detection kit (NCI5079, Thermo Fisher Scientific, Waltham, MA, United States). The data were analyzed using ImageJ software (v 1.52r, Bethesda, MD, United States).

Samples of lung adenocarcinoma participants were gathered for protein detection. RIPA buffer with protease inhibitor cocktail (Roche, Basel, Switzerland) was used for sample preparation and the supernatants were then resuspended in sample buffer. After denaturation of the samples by the way of boiling for 10 min, 30% polyacrylamide gel was used for protein electrophoresis with the same amount (40 μg). Whereafter, PVDF membrane was used for protein transfer (Millipore, Massachusetts, USA). Blocking buffer was used to block membranes for 2 h, then the following antibodies were used SEPT9 (Abcam [Cambridge, USA] ab38314, 1:500), HIST1H2BH (Abcam ab1790, 1:1000); MAPT (Abcam ab64193, 1:500), GAPDH (Goodhere [Hangzhou, China] AB‐P‐R 001, 1:1000), and finally overnight under the condition of 4°C. The membranes were in a state of HRP‐linked secondary antibodies (Boster [Wuhan, China] BA1054, 1:50,000) for 2 h after cleaned by TBST for three times on the following day. An electrochemiluminescence (ECL) detection kit (Thermo [Waltham, USA] NCI5079) was used for immunoreactive protein bands observation at the completion of washing. Azure c300 Gel Imaging System (Azure Biosystems, Dublin, USA) and BandScan software was used to scan and quantify protein bands. Three times have been done for all of the experiments.

Western blot assays were proceeded as previously described [24 (link)]. Briefly, 40 μg total protein was separated by 10% SDS-PAGE and transferred to an activated PVDF membrane (IPVH00010, Millipore, Thermo Scientific, USA). The membrane was immersed in 5% fat-free milk at room temperature for 1 h and incubated with primary antibodies against CysLT1 (ab151484, Abcam, USA), CysLTR1 (a5393, Boster, China), Nrf-2 (ab31163, Abcam, USA), and HO-1 (ab13243, Abcam, USA) at 4°C overnight. β-actin (BM0627, Boster, China) served as the internal control. After incubation with Goat anti-Rabbit IgG Secondary Antibody, HRP (BA1054, BOSTER, China), or Goat anti-Mouse IgG Secondary Antibody, HRP (BA1051, BOSTER, China) at room temperature for 1 h, the membrane was washed with TBST for 3 times. The proteins were visualized after incubating with ECL (NCI5079, Thermo Scientific, USA) using the Imagequant LAS 4000 mini machine (GE Healthcare Life Sciences, USA). All samples were performed at least 3 independent experiments.