Enhanced chemiluminescence western blotting detection kit

The MDCK cell line was grown to approximately 90% confluency in a 6-well plate (1 d), and the cells were infected with H1N1 and incubated for an additional 1 h. After the medium was replaced with DMEM, samples were treated at different concentrations. After 24 h, the cells were washed with chilled PBS and lysed with EBC lysis buffer [1 mM EDTA, 0.5% NP-40, 50 mM NaF, 120 mM NaCl, and 50 mM Tris-HCl (pH 7.6)]. A protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA) was used to measure the protein concentrations. SDS-polyacrylamide electrophoresis was performed using a gel electrophoresis kit. The PVDF membranes (0.45 μm Immobilon-P, Bio-Rad Laboratories, Inc.) to which the proteins were transferred were incubated with the antibodies overnight at 4°C and with the secondary antibodies at room temperature for 1 h in Tris-buffered saline. Finally, the samples were evaluated using an enhanced chemiluminescence Western blotting detection kit (Thermo Fisher Scientific).

Protein content from all samples was quantified using a bicinchoninic acid protein assay kit (23225; Thermo Fisher). Western blot analysis was performed using standard procedures, was detected using an enhanced chemiluminescence western blotting detection kit (32106; Thermo Fisher), and was quantified by scanning densitometry. Antibodies against phosphorylated (p)-protein kinase B (Akt) (Ser473) (#4060), Akt (#4685), p-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204) (#4370), ERK (#4695), VEGF receptor-2 (VEGFR2, #9698), p-VEGFR2 (Tyr1175) (#3770), β-actin (#4970), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, #5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). CD36 (18836), CPT1A (15184), carnitine palmitoyl-transferase 1B (CPT1B; 22170), VEGF (19003), apolipoprotein A1 (APOA1; 14427), and golgi matrix protein 130 (GM130; 11308) were purchased from Proteintech. AGPAT1 (GTX55496) was purchased from GeneTex. CD31 (ab213175), tumor susceptibility 101 (TSG101, ab125011), and CD81 (ab109201) were purchased from Abcam. GAPDH or β-actin was used as a loading control.

Cells were treated with juice powders and flavonoids for 24 h. After treatment, cells were collected and washed with cold PBS. The harvested cells were then lysed in the ice-cold lysis buffer and kept on ice for 30 min. After centrifugation at 13,000 g for 15 min at 4°C, the supernatants were collected, and the protein contents were determined using the Bradford method with bovine serum albumin as the standard. Aliquots of the lysates (40 μg of protein) were boiled for 5 min, and electrophoresis was performed on a 10% sodium dodecyl sulfate-polyacrylamide gel. Proteins in the gels were transferred onto polyvinylidene difluoride membranes, which were then incubated with rabbit polyclonal HO-1 or mouse monoclonal β-actin antibodies (1:1,000 dilutions) at 4°C overnight and then with horseradish peroxidase-conjugated secondary antibody (1:2,000 dilutions) (Cell Signaling, Beverly, MA, USA) for 1 h. Finally, protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Pierce Biotechnology, Rockford, IL, USA). Bands were visualized using a LAS3000® Luminescent image analyzer (Fujifilm, Tokyo, Japan) and quantified by densitometry analysis using PDQuest software (Version 7.0, Bio-Rad Laboratories, Hercules, CA, USA).