Goat anti rabbit igg h l

The BMSCs (mBMSCs and tBMScs) at an initial density of 20,000 cells/well were maintained in the confocal culture dishes for 7 days. Gels containing 10% SDS-PAGE were used to separate equal amounts of total proteins from various BMSC samples at 80 V for 30 min and afterwards at 100 V for 1 h. A PVDF membrane (PerkinElmer, Rodgau, Germany, catalog number: 1620177) was used for protein transfer at 280 mA for 90 min. The cells were then blocked at room temperature for 2 h in the presence of 5% skimmed milk in a trimethylbenzene sulfonyl tetrazole buffer. This was gently shaken at 4 °C overnight with antibodies against TLR4 (1:1000, abcam, Berlin, Germany, catalog number: ab22048), NF-κB p65 Antibody (1:1000, Thermo Fisher Scientific, Dreieich, Germany, catalog number: PA5-27617), p-NF-κB p65 Antibody (1:1000, Thermo Fisher Scientific, Dreieich, Germany, catalog number: PA5-37718), and GAPDH antibody (1:1000, cell signaling technology, MA, USA, catalog number: 2118S). Then, the membranes were incubated with the corresponding secondary antibodies (Goat Anti-Mouse IgG H&L [HRP] [1:5000, Abcam, Berlin, Germany, catalog number: ab6789]; Goat Anti-Rabbit IgG [H + L] [1:5000, Proteintech, Planegg, Germany, catalog number: SA00001-2]) at room temperature for 30 min. As a final step, the immunoreactive protein bands were visualized using enhanced chemiluminescence reagents (ECL Substrate Kit, Abcam, Berlin, Germany, catalog number: ab133406) and examined on an LAS-3000 Imager (FUJIFILM, Tokyo, Japan).

For analysis of protein expression, cells were washed with phosphate-buffered saline (PBS), re-suspended in RIPA buffer (Beyotime), incubated on ice for 30 min, and then centrifuged at 12,000 rpm for 15 min. The supernatant was used to detect the concentration of protein with a BCA Protein Assay kit (Beyotime). The remaining supernatant was added with 5× sodium dodecyl sulfate (SDS, Beyotime) sample buffer and boiled for 10 min. Then, equal amounts of total protein lysates (30–50 μg) were separated by 8–15% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Kankakee, MA, USA). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with the primary antibody at 4°C overnight. After 12- to 16-h incubation, membranes were washed with Tris-buffered saline containing Tween-20 (TBST) buffer and then incubated with secondary antibody. Specific bands were visualized using the enhanced chemiluminescence (ECL) detection kit (Advansta, CA, USA) and analyzed using FluorChem Q system (Protein Simple, CA, USA). The band intensities targeted to proteins were calculated and normalized to that of GAPDH using FluorChem Q system. Three experiments were repeated. The details of antibodies were shown as follows: anti-α-synuclein antibody (Santa Cruz Biotechnology, Santa Cruz, TX, USA), anti-Nurr1 antibody (Abcam, Cambridge, MA, USA), anti-GAPDH antibody (Cell Signaling Technology, Danvers, IL, USA), anti-NF-κB p65 antibody (Millipore), anti-Histone H1.4 antibody (Cell Signaling Technology), Goat anti-mouse IgG H&L (Proteintech Group Inc., Rosemont, IL, USA), Goat anti-rabbit IgG H&L (Proteintech Group Inc.), normal mouse IgG (Millipore), Goat anti-mouse Alexa 594 (Abcam), Goat anti-rabbit Alexa 488 (Cell Signaling Technology), and Hoechst 33342 (Cell Signaling Technology).