Ab254360

The immunohistochemical experiments were conducted as previously described [25 (link)]. The antibodies used in this study included an anti-Iba1 antibody (1:50; ab178846; ab283319; Abcam, MA, USA), an anti-CD86 antibody (1:200; ab220188; Abcam, MA, USA), anti-IL-1β antibody (1:200; ab254360; Abcam, MA, USA), anti-TNF-α antibody (1:150; ab1793; Abcam, MA, USA), anti-IL-6 antibody (1:200; ab290735; Abcam, MA, USA), anti-ERα antibody (1:200; ab32063; Abcam, MA, USA), anti-GPER antibody (1:200; PA5-77396; Invitrogen, Karlsruhe, Germany), anti-ERβ antibody (1:150; PA1-311; Thermo Fisher Scientific, Shanghai, China), anti-NF-κB p65 antibody (1:200; ab32536; Abcam, MA, USA), anti-COX1 antibody (1:500; ab109025; Abcam, MA, USA), anti-CB1 antibody (1:200; ab3558; Abcam, MA, USA), anti-IL-1 (1:200; ab254360; Abcam, MA, USA), Alexa Fluor 488 secondary antibody (1:1,000; Invitrogen, Karlsruhe, Germany), Alexa Fluor 594 secondary antibody (1:1,000; Invitrogen), and IgG H&L (HRP) antibody (1:500; ab97051; Abcam, MA, USA). The nuclei were stained with DAPI (Sigma-Aldrich, Beijing, China) and images were captured on the LSM700 laser scanning confocal microscope (Zeiss, Tokyo, Japan) with the mean fluorescence intensities (MFI) determined using the FlowJo software [26 (link)].

Mouse lung tissue and MLE-12 cells were lysed by RIPA lysate. Mouse lung tissue needs to be additionally put into a homogenizer to fully lyse. The protein lysate was then centrifuged (12,000 g, 15 min, 4°C) and the supernatant was collected. After being mixed into the loading buffer, the protein lysate was heated to 100°C for 5 min. Then, we made a 10% sodium dodecyl sulfate polyacrylamide gel and added 10 μL of protein lysate. After electrophoresis and membrane transfer, the protein is transferred to the polyvinylidene fluoride membrane. We used 5% bovine serum albumin-PBS-Tween to block nonspecific antigens for 1 h at room temperature. The membrane was then incubated with primary antibody dilutions (β-actin, ab8226, Abcam, Cambridge, MA, USA; IL-1β, ab254360, Abcam, Cambridge, MA, USA; tumor necrosis factor (TNF), ab1793, Abcam, Cambridge, MA, USA; SOD1, ab51254, Abcam, Cambridge, MA, USA; SOD2, ab68155, Abcam, Cambridge, MA, USA; caspase 3, ab184787, Abcam, Cambridge, MA, USA; p65, ab16502, Abcam, Cambridge, MA, USA; p-p65, ab86299, Abcam, Cambridge, MA, USA; IκBα, ab183503, Abcam, Cambridge, MA, USA;) at 4°C overnight. After washing the membrane, we incubated the membrane at room temperature for 1 h with the secondary antibody dilution (Abcam, Cambridge, MA, USA). Finally, we used chemiluminescent liquid for color development.

Proteins in the brain tissues and lung tissues were harvested by a RIPA buffer (P0013D, Beyotime, China) and their concentrations were qualified with a BCA Kit (pc0020, Solarbio, China). After denaturation, the protein samples were separated by electrophoresis. Proteins in the gel were transferred to a nitrocellulose membrane (10600023, GE Healthcare Life, USA), which was then sealed a 5% skim-milk. After that, they were reacted with primary antibodies at 4 °C overnight. After washing, they were reacted with anti-rabbit HRP (1:5000, #7074, CST, USA) at 37 °C for 1 h. In the end, the protein signals were developed by the ECL reagent (35055, Pierce, USA) in a gel imaging system (A44114, Invitrogen, USA). The primary antibodies of TNF-α (ab205587, 1:1000), IL-1β (ab254360, 1:1000), p-NF-κB (ab76302, 1:1000), NF-κB (1:5000, ab32536), p-IKBα (1:10,000, ab133462), IKBα (1:5000, ab32518), HIF-1α (1:1000, ab179483), and GAPDH (1:5000, ab199554) were obtained from Abcam (UK).