Ab195049

Radioimmunoprecipitation Assay Lysis Buffer (Beyotime, Nanjing, China) was used to extract proteins from microglial cells. Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was applied to determine the concentration of isolated proteins. Protein samples were loaded at 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore, USA). Subsequently, the membranes were incubated with primary antibodies at 4°C overnight, which included anti-Map3k12 (JK221305, Shanghai JingKang Bioengineering CO., LTD., Shanghai, China), anti-BACE1 (ab183612, 1:1000, Abcam), anti-Tau-5 (ab80579, 1:1000, Abcam), anti-p-ERK1/2 (ab278538, 1:100, Abcam), anti-ERK1/2 (ab17942, 1:1000, Abcam), anti-p-p38 (ab195049, 1:1000, Abcam), anti-p38 (ab31828, 1:1000, Abcam), and GAPDH (ab8245, 1:1000, Abcam). Then, the membranes were incubated with secondary antibodies at room temperature for 2 h. The protein bands were imaged by enhanced chemiluminescence reagent (Bio-Rad) and analyzed by ImageJ software (National Institutes of Health, Bethesda, MA, USA) [40 (link)].

Lung tissues (50 mg per mouse) were homogenized in ice‐cold RIPA buffer (Sigma‐Aldrich) with a 1% protease inhibitor cocktail and a phosphatase inhibitor, and protein concentration was examined using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Thereafter, proteins (40 μg) were separated by 10% SDS‐PAGE and blotted onto a PVDF membrane. After blocking with 5% skimmed milk for 2 h, the membrane was incubated overnight with primary antibodies against p‐JAK2 (ab32101, 1:4000; Abcam), JAK2 (ab108596, 1:5000; Abcam), phosphorylated STAT3 (ab76315, 1:5000; Abcam), STAT3 (ab68153, 1:2000; Abcam), SOCS3 (ab280884, 1:1000; Abcam), p‐NF‐κB p65 (ab76302, 1:1000; Abcam), NF‐κB p65 (ab32536, 1:1000; Abcam), p‐p38 MAPK (ab195049, 1:1000; Abcam), p38 MAPK (ab170099, 1:1000; Abcam), and GAPDH (ab9485, 1:2500; Abcam) at 4°C, and subsequently incubated with secondary antibodies for 2 h at room temperature. The bands were visualized by enhanced chemiluminescence reagent (Beyotime), and the intensity of blot was quantified by Image Lab 3.0 software (Bio‐Rad, Hercules, CA, USA).

The PTC tissues and cells lysates were collected using RIPA buffer (Invitrogen, shanghai, China) and protease inhibitor cocktail (#5871; Beverly, MA, USA). Protein concentrations were calculated using Bicinchoninic Acid Kit (Takara; Merck KGaA) referring to the manufacturer’s protocols. Immunoblotting procedures followed the previously published method [13 ]. Equal amounts (60 µg) of total tissues or cellular proteins were added into loading buffer, boiled for 5 min, and separated on 10 % SDS-PAGE electrophoresis. Subsequently, the proteins were metastasized to nitrocellulose (NC) membranes. Then we blocked the membranes with 5 % non-fat dry milk for 1 h at room temperature and hatched overnight with primary antibodies. Following further incubated with secondary antibody for 1 h, the immunoreactive protein strips were visualized by ECL kit (Thermo, CA, USA) on the Bio-Rad Chemical Doc XRS system (Bio-Rad). The primary antibodies were including anti-p-p38 (ab195049; 1:2000), anti-p38 (ab250612; 1:1000), anti‐p-JAK2 (ab32101; 1:1500), anti‐JAK2 (ab39636; 1:800), anti‐p-STAT1 (ab215820; 1:1000), anti‐STAT1 (ab155933; 1:1000) and anti‐β‐actin (ab179467; 1:5000) were purchased from Abcam.

Cell lysate was collected using RIPA lysis buffer. The cell protein mass of each lysate was determined by BCA Protein Assay Reagent (Pierce, IL). Proteins were separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to membranes (0.22 μm, Sigma) and incubated with primary antibodies at 4°C overnight. The primary antibodies are as follows: antibodies against VEGFA (1/1000; ab1316, Abcam), TGFβ1 (1/1000; ab215715, Abcam), Angiopoietin-1 (1/20000; ab183701, Abcam), ZO-1 (1/1000; ab216880, Abcam), Occludin (1/1000; ab216327, Abcam), Claudin-5 (1/1000; ab131259, Abcam), G3BP2 (1/2000; ab86135, Abcam), p-p38 (1/1000; ab195049, Abcam), p38 (1/2000; ab170099, Abcam), p-p53 (1/2000; ab33889, Abcam), p53 (1/1000; ab26, Abcam), and GAPDH (1/500; ab8245, Abcam). Next, the membranes were incubated with HRP-conjugated secondary antibody IgG (1/2000; ab7090, Abcam) at room temperature for 2 h. GAPDH antibody served as a negative control. At last, the protein bands were visualized by an ECL Western Blotting Substrate Kit (ab65623, Abcam) and quantified by the ImageJ software.

Collected infarcted cortex tissue of rats was sonicated in the homogenization buffer (RIPA with protease cocktail inhibitor, phosphatase inhibitor, and phenylmethanesulfonyl fluoride). Protein concentration was determined using the bicinchoninic acid assay. Equal amounts of the samples and sample buffer were mixed and boiled for 10 min at 95°C and were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gel, and the proteins were transferred to a PVDF membrane (Millipore, PA, USA). The membrane was then incubated with the following primary antibodies overnight at 4℃: p38 (1:1,000, Abcam, ab170099), p-p38 (1:1,000, Abcam, ab195049), JNK (1:1,000, Abcam, ab179461), p-JNK (1:1,000, Abcam, ab124956), ERK1/2 (1:1,000, Abcam, ab184699), p-ERK1/2 (1:1,000, CST, 4370T), Iba-1 (1:1,000, Abcam, ab178847), iNOS (1: 1,000; Abcam, ab178945), SOD1 (1:1,000; Abcam, ab51254), Bax (1:500, Proteintech, 50599-2-Ig), and Bcl-2 (1:500, Proteintech, 12789-1-AP). After washing with TBST three times, the membrane was incubated with horseradish peroxide-conjugated secondary antibodies for 60 min at room temperature. The chemiluminescence signals were captured using EPSON Imaging System (EPSON; V300, Japan). Densitometric analysis was performed using Alpha Software (Alpha Innotech; alphaEaseFC, USA). β-Actin was used as a loading control.