Polyvinylidene fluoride blotting membrane

Exosomes were analyzed using negative-staining transmission electron microscopy (TEM). The exosomes were suspended in 2% glutaraldehyde and loaded onto copper grids, after which they were negatively stained with 3% (w/v) aqueous phosphotungstic acid for 1 min. The grids were examined using the FEI Tecnai G2 Spirit Twin transmission electron microscope. In addition, macrophage exosomes were analyzed using the NanoSight NS300 instrument (Malvern Instruments, UK).
The markers CD63, CD9, CD81, CCL1 of exosomes and CCL1, CCR8, and IL-4 of the mice colon were detected by western blotting. The standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) procedure was followed, macrophage exosomes were lysed in a western blotting lysis buffer and separated using 10% SDS-PAGE. Subsequently, proteins were electrophoretically transferred onto a polyvinylidene fluoride blotting membrane (GE Healthcare Life Sciences, UK), and then blocked using 5% skimmed milk. The membranes were incubated with anti-mouse CD63, CD9, CD81, CCL1, CCR8, and IL-4 antibody (BD Biosciences, USA) overnight at 4°C. Anti-mouse antibodies conjugated with horseradish peroxidase (HRP) (Jackson Immunoresearch Labs Inc., USA) were used as secondary antibodies. Membranes were visualized via an enhanced chemiluminescence (ECL) chemiluminescent detection system (Amersham, USA).

Cells and liver tissues were homogenized using RIPA lysis buffer in the presence of freshly added protease and phosphatase inhibitors (Thermo Fisher Scientific, USA). Lysates were then quantified and subjected to 10% sodium dodecyl-polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene fluoride blotting membrane (GE Healthcare Life Sciences, UK). The membranes were blocked using 5% skim milk and then incubated with IL-10 (1:500; Proteintech, China) and PPAR-γ (1:1000; Proteintech, China, 60,269-1-Ig) antibodies. GAPDH (1:5000; Proteintech, China, 60,004-1-Ig) and β-actin (1:5000; Proteintech, China, 66,009-1-1 g) were used as internal standards. The membranes were visualized using an ECL western blot detection system (Amersham, USA).

PCa cell lines were lysed using radioimmunoprecipitation assay buffer (Catalog No. 89900, Thermo Fisher Scientific, Waltham, MA). Protein concentrations were measured by BCA assay (Catalog No. 23224, Thermo Fisher Scientific). Samples were separated via 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) for 90 min and transferred to polyvinylidene fluoride blotting membranes (0.2 μm pore size; GE Healthcare, Chalfont St. Giles, UK) for 2 h. The membrane was blocked in 5% bovine serum albumin for 2 h. Primary antibodies against SIRT1 (Catalog No. 8469S, Cell Signaling Technology Technology, Beverly, MA), SIRT2 (Catalog No. 12672S, Cell Signaling Technology), SIRT3 (Catalog No. 5490S, Cell Signaling Technology), SIRT4 (Catalog No. ab231137, Abcam, Cambridge, UK), SIRT5 (Catalog No. 8782S, Cell Signaling Technology), SIRT6 (Catalog No. 12486S, Cell Signaling Technology), SIRT7 (Catalog No. 5360S, Cell Signaling Technology) and succinyl-lysine (PTM Biolabs, Chicago, IL) were used to capture SIRT1-SIRT7 proteins overnight at 4 °C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, the membranes were washed in Tris-buffered saline-0.1% Tween 20 (TBS-T) three times for 15 min. ECL prime Western blotting detection reagent (RPN2232, Citiva) was used to visualize the blots.

Western blotting was performed as described previously (Hishiki et al., 2014 (link)). Briefly, DENV-1-infected A549 cells were co-cultured with 10 μM hirsutine, 100 μM ribavirin, or 10 μM bromocriptine. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride blotting membranes (GE Healthcare, Little Chalfont, United Kingdom). Anti-NS3 rabbit polyclonal antibodies (GTX124252; GeneTex, Irvine, CA, United States) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibodies (MAB374; Millipore, Billerica, MA, United States) were used as primary antibodies.

Liver tissues, neutrophils, and macrophages were homogenized using RIPA lysis buffer in the presence of freshly added phosphatase and protease inhibitors (Thermo Fisher Scientific, USA). Lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred onto polyvinylidene fluoride blotting membranes (GE Healthcare Life Sciences, UK) and blocked using 5% skim milk. The membranes were incubated with the following primary antibodies (overnight at 4°C): anti-mouse N-WASP (1:1,000; Abcam), IL-10 (1:500; Proteintech, China), CCL2 (1:500; Boster, China), α-SMA (1:1,000; Abcam), collagen I (1:2,000; Proteintech), and TNF-α (1:1,000; Abcam). GAPDH (1:5,000; Sigma-Aldrich, USA) and β-actin (1:5,000; Sigma-Aldrich) were used as internal standards. Blots were incubated with anti-IgG conjugated to horseradish peroxidase as the secondary antibody for 2 h at room temperature. The membranes were visualized using an ECL western blotting detection system (Amersham, USA).