Odyssey v 3.0 image scanner

PAX6 antibody (9013101, Biolegend, San Diego, CA, USA) was generated against only p46 and p48 PAX6. GAPDH antibody was from Abcam (ab8226, Abcam, MA, USA). Procedures and other antibodies were as previously described^{34 (link)}. Bound antibodies were detected by DyLightTM680-conjugated secondary antibodies (Sigma, St. Louis, MO, USA), and the protein expression levels were observed using an Odyssey V 3.0 image scanner (LI-COR). These blots were repeated at least three times for each condition.

Total protein was extracted from tumor tissue and quantified with a BCA protein quantification kit (Beyotime Biyuntian Biotechnology Co., Ltd., China, Cat. No. P0010). A total of 10 µg of each protein sample was separated by SDS-PAGE and then transferred to a PVDF membrane, which was incubated with primary antibodies (P-gp: Absin, rabbit, 1:1000; VEGF: Abcam, ab46154, 1:1000; and GAPDH: 1:10000) at 4 °C overnight and then with secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit, ASPEN, AS1107, 1:10000) for 1 h at room temperature. Antibody reactivity was detected with an Odyssey V3.0 image scanner (Li-COR. Inc., Lincoln, NE, USA). Quantitative analysis of band intensity was conducted with ImageJ.

After RPE cells were incubated with either the miR-29a mimics or inhibitor for 12 hours, total cellular proteins were harvested for western blot analyses. A BCA kit (Pierce, Rockford, IL, USA) was used to determine the protein concentrations. A 40 μg protein sample was then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the proteins. Subsequently, the proteins were transferred to a 0.22 mm polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% bovine serum albumin and incubated with rabbit monoclonal anti-MMP-2 at a dilution of 1 : 300 (SAB, MD, USA), followed by mouse anti-β-actin at a dilution of 1 : 5,000 (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. After incubation with DyLight™ 680-conjugated secondary antibodies at a dilution of 1 : 5,000 (Sigma-Aldrich), the protein expression levels were determined using the Odyssey V 3.0 image scanner (LI-COR, Lincoln, NE, USA). The intensities of the protein bands were quantified densitometrically using BandScan software, ver. 5.0, and the values for each sample were normalized against β-actin.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and proteins were extracted. BCA protein assay kit was used to determine the concentrations of protein samples. Then protein samples were separated by 10% SDS-PAGE and transferred to NC membrane. Next, the membrane was blocked and incubated in primary antibody at 4°Cfor 8 h (p-JAK, JAK1, p-STAT3, STAT3, SOCS3, and β-actin at 1:1000, 1:1000, 1:1000, 1:1000, 1:1000, and 1:5000, respectively). Then the membrane was incubated in secondary antibody (1:10,000) for 90 min after washed by TBST for four times. At last, the protein expression was detected by ECL chemiluminescence buffer and tested by an Odyssey v3.0 image scanner (LI-COR Biosciences, NE, USA). Band was analyzed by Image J software.

Murine osteoclast precursor RAW264.7 cells were pretreated with 5 μM plumbagin for 6 h, followed by stimulation with 50 ng/ml RANKL for 0, 5, 10, 20, 30, or 60 min. Cells were then lysed with RIPA buffer (Beyotime, Shanghai, China) for protein extraction. Equal volumes of protein lysates were resolved using SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States). These membranes were incubated with the appropriate primary antibodies (Cell Signaling Technology, Danvers, MA, United States) at 4°C overnight before secondary antibody incubation (Abcam, Cambridge, United Kingdom) for 2 h at room temperature. The results were detected with an Odyssey V3.0 image scanner (Li-COR. Inc., Lincoln, NE, United States). The experiments were repeated independently for at least three times.