Hrp conjugated secondary antibody

The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x10 4 cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China).
2.9 Western blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).

The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x10 4 cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China). 2.10 Protein blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).

The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x10 4 cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China).
2.10 Protein blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).

Total proteins were extracted from the paw of CIA mice and their concentration was determined using a BCA assay (Beyotime, Nanjing, CHN). Protein samples were applied and separated on 10% NuPAGE gel (Invitrogen, New York, NY), followed by transferring onto PVDF membranes (Millipore Inc., Darmstadt, Germany). Membranes were blocked in 5% non-fat dry milk and 0.1% Tween-20 for 1 h, followed by incubation overnight with primary antibody against mouse NF-κB p50 (Abcam, Cambridge, UK, ab32360), NF-κB p65 (Cell signaling, Danvers, MA, #8242T), Act 1 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-100647), TRAF6 (Immunoway, Suzhou, CHN, YT4720), IL-17RA (Absin Bioscience, Shanghai, CHN, abs140681), and GAPDH (Absin Bioscience, Shanghai, CHN, abs100005) diluted at 1:1000 in blocking solution. Next, the HRP-conjugated secondary antibody (Absin Bioscience, Shanghai, CHN) at a dilution of 1:8,000 was used to incubate the membranes for 2 h. Immunoreactive proteins were visualized using Tanon high-sig ECL Western blotting Substrate and Tanon 5200 multifunction laser-scanning system (Tanon, CHN).

2.5 × 10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).