Gel doc ez imager

The total protein of aortic tissues in each group was extracted. The protein concentration was determined according to the bicinchoninic acid protein assay kit (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The extracted protein added with uploading buffer and separated with 10% polyacrylamide gel electrophoresis (Wuhan Boster Biological Technology Co., Ltd., Wuhan, Hubei, China). The proteins were transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin (BSA) for 1 h. Primary antibodies of Caspase-3, Bax, Bcl-2, VEGF, PI3K, p-PI3K, AKT, p-AKT and β-actin (1: 3000, Abcam, Cambridge, MA) were added and incubated at 4 °C overnight, followed by washing three times (5 min per wash) with Tris-buffered saline with Tween 20 (TBST). Corresponding secondary antibodies (Shanghai Miaotong Biotechnology Co., Ltd., Shanghai, China) were added and incubated for 1 h. The membranes were washed for three times with 5 min for each time. Chemiluminescence reagents were employed to develop images. β-actin was considered as an internal reference. The images of the gels were captured in a Bio-Rad Gel Doc EZ Imager (Bio-Rad, Hercules, CA). The gray values of target protein bands were analyzed by ImageJ software (National Institutes of Health, Bethesda, MA). The experiment was conducted in triplicate.

Total protein extraction from NR8383 AM cells (5 × 10⁶ cells) and rat lungs were performed with radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher Scientific, Shanghai City, China). We used 10% SDS-polyacrylamide gel electrophoresis to separate the protein samples and transferred them to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Next, the membranes were blocked with 5% non-fat milk for 1 h at room temperature (RT) and incubated with primary antibodies for 12 h at 4 °C. Then, the membranes were incubated with secondary antibodies at RT for 4 h. We used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal reference. All blots were visualized by the enhanced chemiluminescence kit assay (Millipore, Billerica, MA, USA) and were developed with a Bio-Rad Gel Doc EZ imager (Bio-Rad, USA), and the band intensities were analysed by ImageJ software (NIH Image analysis). The primary antibodies used in this experiment included rabbit polychonal anti-NLRP3 (1 : 500; Cell NBP2-12446, Novus, USA), anti-GAPDH (1 : 2000; Cell Signaling Technology, USA), and anti-caspase-1 (1 : 2000, ab1872, Abcam, USA).

Total protein in tissues and cells was extracted and protein concentration was determined via bicinchoninic acid kits (BOSTER Biological Technology Co., Ltd., Hubei, China). The extracted proteins were conducted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BOSTER Biological Technology) and transferred onto polyvinylidene fluoride membranes, which were blocked with 5% bovine serum albumin for 1 h. Then, the membranes were incubated with primary antibodies DACT2 (1:1000), active-β-catenin (1:5000), Glut1 (1:1000), β-actin (1:1000, all from Abcam Inc., MA, USA), p-β-catenin (1:1000), and lactate dehydrogenase A (LDH-A, 1:1000, both from Cell Signaling Technology, MA, USA) at 4 °C overnight. Next, the membranes were incubated with relative secondary antibody (Shanghai Miaotong Biotech Co., Ltd., Shanghai, China) for 1 h and developed using enhanced chemiluminescent reagent and the Bio-Rad Gel Doc EZ imager (Bio-Rad Laboratories, Hercules, CA, USA). Image J software (National Institutes of Health, Bethesda, Maryland, USA) was employed to analyze the gray values of the protein bands.

Sodium Dodecyl Sulfate (SDS)-PAGE was performed for pure LcrV and LcrV formulation PD1 (stored at RT) using 12% w/v, acrylamide separating gel, and a 4% w/v, stacking gel both containing 0.1% w/v, SDS [18 (link)]. Non-denaturing polyacrylamide gel electrophoresis was also performed [19 (link)]. Following Coomassie brilliant blue staining, the gels were photographed with Bio-Rad Gel Doc EZ imager (Bio-Rad Laboratories, Hercules, CA, USA), and the photographs were analyzed and annotated using Image lab 4.1 software (Bio-Rad Laboratories, Hercules, CA, USA) to determine each protein band.

SDS-PAGE of the enzymes was performed according to the method of Laemmli [26] (link) using 10% gel. The molecular weight of the enzyme(s) was determined by using a medium range protein molecular weight marker (97.4–14.3 kDa, GeNei, Bangalore). A zymogram of CaCl₂ treated crude enzyme was accomplished on separating gel, containing 0.1% gelatin as the substrate. Following electrophoresis, the gel was washed successively 2.5% (v/v) triton X-100 twice to remove SDS. The gel was then incubated with developing solution (Tris buffer, pH 7.4) at 37 °C for 18 h. Later, the gel was stained with coomassie brilliant blue R250 for 30 min and destained overnight to reveal zones of substrate lysis. The intensity of the zones was measured using BIO RAD Gel Doc EZ Imager.

Total proteins in tissues, cells and exosomes were extracted by radio-immunoprecipitation assay lysis buffer (R0010, Solarbio Science & Technology Co. (Beijing, China). The protein concentration was determined by bicinchoninic acid kit (Shanghai Yanxi Biotechnology Co., Ltd., Shanghai, China). The abstracted protein was appended to the loading buffer, boiled at 95 °C for 10 min (30 μg/well), and isolated with 10% sodium dodecyl sulfate polyacrylamide gel electropheresis. The protein was transferred to a polyvinylidene fluoride membrane by a semidry electrophoretic transfer apparatus (Sigma-Aldrich, SF, CA, USA) and sealed with 5% bovine serum albumin (AmyJet Scientific Inc., Wuhan, Hubei, China). The primary antibody LRRC1 (1:500), P-gp (1:500), TopoIIα (1:10000), GST-π (1:1000, Abcam, Cambridge, MA, USA), CD63 (1:100, BD Biosciences, Lake Franklin, New Jersey, USA), and CD81 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were appended. The horseradish peroxide-conjugated secondary antibody (1:1000, AmyJet Scientific Inc., Wuhan, China) was incubated for 1 h. The image was developed by chemiluminescence reagent. GADPH (1:10,000, Abcam) was utilized as a loading control. Bio-rad Gel Doc EZ imager (Bio-Rad, California, USA) was utilized to development while Image J software (National Institutes of Health, Bethesda, MD, USA) to protein band evaluation.