Radio immunoprecipitation assay ripa buffer

Scopolamine hydrobromide, sodium nitrite (NaNO2), Griess reagent, dimethyl sulfoxide (DMSO), dichlorofluorescin diacetate (DCFDA), phosphate buffer, DPPH, donepezil, and ascorbic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-ChAT and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from EMD Millipore (Billerica, MA, USA). Anti-AChE was obtained from Abcam (Cambridge, MA, USA). Radioimmunoprecipitation assay (RIPA) buffer was obtained from Thermo Scientific (Waltham, MA, USA). All horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA). Chebulic acid, gallic acid, corilagin, chebulanin, 1,3,6-tri-O-galloyl β-D-glucose, chebulagic acid, and chebulinic acid were provided by Professor Sang Hyun Sung (Seoul National University, College of Pharmacy, South Korea). The purity of all reference chemical substances was higher than 95%. HPLC-grade water and acetonitrile were purchased from J.T. Baker (Phillipsburg, NJ, USA).

Two μg each of anti-L1 cell-adhesion molecule (L1CAM, clone 5G3, Santa Cruz Biotechnology) for enrichment of neuronal exosomes, anti-myelin oligodendrocyte glycoprotein (MOG, clone D-2, Santa Cruz Biotechnology) for enrichment of oligodendroglial exosomes, or normal mouse IgG (Life Technologies) as a negative control were used to coat 1 mg of M-270 epoxy Dynabeads using a Dynabeads Antibody Coupling Kit (Life Technologies) overnight at 37 °C with gentle rotation following the manufacturer’s instructions. Antibody-coated beads then were mixed gently with the isolated serum/plasma EVs in chilled phosphate-buffered saline (PBS), pH 7.4, containing 1% (w/v) BSA and PPi and incubated overnight at 4 °C with gentle rotation. The bead-attached exosomes then were washed with 1 mL of 0.1% (w/v) BSA in PBS, pH 7.4, and transferred into new tubes in which the exosomes were lysed by incubating in 25 μL of radioimmunoprecipitation assay (RIPA) buffer (Thermo-Fisher Scientific) containing PPi for 10 min at room temperature and stored at − 80 °C.

The Dimethylmethylene Blue (DMMB) assay was used to quantify total glycosaminoglycan (GAG) amount in each material at 7 days and 21 days of culture. A stock solution of dimethylmethylene blue (DMMB) was made by dissolving 32 mg of 1,9-DMMB in 20 mL of pure ethanol overnight on an orbital shaker at room temperature. This stock solution was added to a mixture of 1.5 L distilled water, 59 mL 1 M NaOH and 7 mL 98% formic acid and left to mix for 2 h. The pH of the dye was adjusted and verified to be pH 1.5 prior to use. The cell-laden biomaterial hemispheres of each bioink were lysed in Radioimmunoprecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, MA, USA) to yield protein isolates for GAG quantification along with material only control samples. Isolates were diluted 1 in 50 with distilled water and 40 μL added to the wells of a 96 well plate in triplicate with 200 μL DMMB reagent per well. The plates were read immediately at 525 nm compared to a series of chondroitin standards ranging from 0 to 50 μg/mL (0.03–0.75 μg). The total content of glycosaminoglycan in each sample was acquired using the standard curve of chondroitin samples, corrected for sample volume and dilution.

Monoclonal antibodies against PKM2, STAT1, p-STAT1, pro-caspase-3, cleaved caspase-3, and Mcl-1 were purchased from Cell Signaling Technology (Boston, MA, USA).
Cells were collected, washed with PBS and placed into 1 × Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Waltham, MA, USA). After lysing the cells for 45 min on ice, supernatants were collected through centrifugation at 16,000 × g, 4 °C for 10 min and boiled for 10 min with loading buffer (Epienzyme, Shanghai, China). Total protein (25 μg), as measured by the bicinchoninic acid method, was separated on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes of 0.45 µm pore size. The membranes were blocked and incubated with primary antibodies diluted at a ratio of 1:1000 overnight at 4 °C. Next, the membranes were washed with 1 × Tris-buffered saline with Tween 20 (TBST) 3 times and incubated with horseradish peroxidase (HRP)-linked secondary antibodies for 1 h at room temperature. Specific bands were observed by enhanced chemiluminescence (ECL).

VSMCs after different treatment lysed with Radio-Immunoprecipitation Assay (RIPA) buffer (Thermo Fisher Scientific), then the concentration of protein was monitored with a protein assay Kit (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of protein lysates were loaded and separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were firstly blocked in 5% non-fat milk for 1 h at room temperature, and incubated with primary antibodies against PCDA (1:1000 dilution), Ki-67 (1:1000 dilution), LDHA (1:1000 dilution), and GAPDH (1:2000 dilution). Then the membranes were incubated with corresponding HRP-conjugated secondary antibodies (1:2000 dilution) at room temperature for 2 h. Protein bands were visualized using an Enhanced Chemiluminescence Kit (Bio-Rad, Hercules, CA, USA), detected using ChemiDoc (Bio-Rad) and analyzed by densitometry (Image Lab, Bio-Rad).All antibodies were purchased Cell Signaling Technology (Danvers, MA, USA).