0.2 μm pvdf membrane

Tissue sample homogenates were treated with RIPA lysis buffer containing protease and phosphatase inhibitors (cOmplete™, Mini Protease Inhibitor Cocktail, Roche, Switzerland, PhosSTOP™, Roche, Switzerland). Protein concentration in the filtrate was determined with the BCA method using a commercial Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific™, Waltham, MA, USA). Then, electrophoretic protein fractionation was conducted (SDS-PAGE) using 10% polyacrylamide gel by placing 30 μg protein/well. The fractionated proteins were transferred onto a 0.2 μm PVDF membrane (Thermo Fisher Scientific™, Waltham, MA, USA) by a wet transfer. Prior to incubation with antibodies, the membranes were placed in a blocking buffer (5% BSA) for 60 min. The brain protein expression was detected using an antibody against D1R (ab81296) and D2R (ab85367)—rabbit pAb (Abcam, Cambridge, UK) diluted 1:800 and 1:1000, respectively, and sAb goat anti-rabbit IgG HRP H&L (ab97051) (Abcam, Cambridge, UK). Expression of β-actin was detected using the β-Actin antibody: sc-47778 (Santa Cruz Biotechnology, US). The membranes were developed with an ECL Advance Western Blotting Detection Kit (GE Healthcare, Chicago, IL, USA), and subsequently, bands were visualized using the Molecular Imager ChemiDock XRS+ (Bio-Rad, Hercules, CA, USA).

Cells were washed once with ice‐cold PBS and lysed in 60 μl RIPA Lysis Buffer (Thermo Fisher Scientific) supplied with 1% protease inhibitor cocktail set III (Merck Chemicals) for 30 min at 4°C. Cell debris was pelleted for 10 min at 13,000 g and 4°C, and the supernatant was transferred to a fresh tube. Protein concentration was determined with Thermo Scientific's Pierce™ BCA protein assay kit according to the manufacturer's instructions. Protein lysates were mixed with 4 X NuPAGE LDS Sample Buffer (Invitrogen), supplied with 10% 2‐mercaptoethanol (Roth), and inactivated for 10 min at 99°C. Proteins were separated by size on a 12% sodium dodecyl sulfate polyacrylamide gel and blotted onto a 0.2 μm PVDF membrane (Thermo Scientific) by semi‐dry blotting (BioRad). Human ACE2 was detected using a polyclonal goat anti‐human ACE2 antibody (1:500, R&D Systems), a horseradish peroxidase (HRP)‐labeled donkey anti‐goat antibody (1:5,000, Dinova), and Super Signal West Femto Chemiluminescence Substrate (Thermo Fisher Scientific). As a loading control, samples were analyzed for β‐Actin expression using a mouse anti‐β‐actin antibody (1:5,000, Sigma Aldrich) and an HRP‐labeled goat anti‐mouse antibody (1:10,000, Dianova).

Raw264.7/effluc cells were treated with or without DEX for 1 h and then with 1 μg/mL LPS for 24 h. They were washed twice with cold PBS and lysed with RIPA buffer containing a complete protease inhibitor cocktail and phosphate inhibitor (Roche, Basel, Switzerland). Equal amounts of proteins were loaded in each lane and resolved by 4%–12% gradient Bis-Tris gels (Invitrogen, Calrlsbad, CA, USA). Proteins were transferred onto 0.2 μm PVDF membranes (Invitrogen, Calrlsbad, CA, USA). The membranes were incubated overnight at 4 °C with the following primary antibodies: COX-2 (Cayman; dilution 1:3000), p-AKT (Cell Signaling, Danvers, MA, USA; dilution 1:1000), p-ERKphospho-p44/42 MAPK (phosphorylated extracellular-signal-regulated kinase (pERK1/2)) (Cell Signaling; dilution 1:2000), β-actin (Cell Signaling; dilution 1:3000). The membranes incubated with the primary antibody were then incubated with an HRP-conjugated secondary antibody at room temperature. ECL-Plus (Millipore, Burlington, MA, USA) was used to detect peroxidase activity according to the manufacturer’s protocol.
Murine macrophage Raw264.7 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA) at 37 °C in a 5% CO₂ atmosphere. The establishment of Raw264.7/effluc cells was described previously [29 (link)].

Cell pellets were lysed in radioimmune precipitation assay (RIPA) buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 0.1% SDS, 1 × Protease inhibitor cocktail set 1 (Calbiochem; La Jolla, CA). Protein concentration was determined with the bicinchoninic acid BCA assay (Pierce from Thermo Fisher Scientific; Waltham, MA), and equal amounts of proteins (15 μg) were separated by NuPAGE®Novex® 4–12% Bis-Tris protein gels using NuPAGE®MES SDS Running Buffer (Life Technologies; Grand Island, NY). Proteins then were transferred onto 0.2 μm PVDF membranes (Invitrogen; Waltham, MA), which were subsequently blocked with 5% nonfat milk for 1 h and probed with the indicated antibodies (AXL, 1:1000, CST; pAXL, 1:1000, CST; pSTAT3, 1:1000; STAT3, 1:1000; pERK, 1:1000, CST; ERK, 1:1000, CST; pAKT, 1:1000, CST; AKT, 1:1000, CST; Actin 1:3000, CST; PD-L1, 1:1000, Abcam, HSP27, 1:1000, CST; pHSP27, 1:1000, CST; AMPK, 1:1000, CST; pAMPK, 1:1000, CST), and then incubated with corresponding HRP-linked secondary antibody (1:2000, CST, CA, USA) at 4°C over-night. Membranes were then washed, and blots were developed with the enhanced western Chemiluminescent HRP Substrate (Pierce from Thermo Fisher Scientific; Waltham, MA) for 2–3 min, and then photographed using a Bio-Rad Gel Doc XR and Imaging System.

Protein lysates from cell and tissues were lysed and homogenized (tissues) in Mammalian Protein Extraction Reagent (M-PER) lysis buffer (Cat. no. 78501; Thermo Fisher Scientific) supplemented with cOmplete protease inhibitor tablets (Cat. no. 1183617000; MilliporeSigma). Approximately 50 μg of lysate were electrophoretically resolved on 4–20% Novex Tris-glycine gradient gels (Cat. no. XV04200PK20; Thermo Fisher Scientific). Proteins were then transferred to 0.2-μm PVDF membranes (Cat. no. 88520; Thermo Fisher Scientific) and incubated overnight at 4°C with gentle rocking in primary antisera diluted 1:1,000 in 5% bovine serum albumin (Cat. no. A30075; RPI Corp)/1xTBS-Tween (Cat. no. IBB-581X; Boston BioProducts). Membranes were washed three times in 1xTBS-Tween for 5 min each and then incubated in secondary antisera (1:2,000 dilution) for 1 h at room temperature. Membranes were then washed four times in 1× TBS-Tween for 15 min each before the addition of the Novex Chemiluminescent Substrate Reagent Kit (Cat. no. WP20005; Thermo Fisher Scientific). Membranes were exposed onto PerfectFilm audioradiography film (Cat. no. B581; GenHunter) and developed on a Typhoon Variable Mode Imager (Amersham Pharmacia). Western blot densitometry was performed using open-source ImageJ software.