Millipore ecl kit

The NP cells were washed three times with cold phosphate-buffered saline (PBS) and cell lysates were extracted on ice. The total protein concentration of different lysates was determined according to the Bio-Rad Bradford Protein Assays kit (ThermoFisher) using bovine serum albumin (BSA) as a standard. Equal amounts (40 μg protein per lane) of proteins were separated on analytical 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidenedifluoride membrane (PerkinElmer) by a semidry apparatus. Primary antibodies against the following proteins were used: cleaved form PARP, aggrecan, IL-1β, IL-8, TNF-α, p-ERK1/2, ERK2, p65, p-p65 (ser536), p-p38, p38, p-JNK1/2/3, JNK, TLR2, MyD88, and β-Actin were employed as primary antibodies. Immunoblot analysis was performed using secondary mouse or rabbit immunoglobulin G (IgG) antibody coupled with horseradish peroxidase and detected by the Millipore ECL kit (Thermo Fisher Scientific, UK). The densities of the immunoblots were determined using an image analysis system installed with a software BIO-ID (VilberLourmat, France).

For Western blots of whole-cell lysates, cell pellets were dissolved directly into SDS loading buffer. For cell fractions, 5× SDS loading buffer was added. In both cases, samples were boiled and resolved by SDS-PAGE. Gels were transferred to 0.45 μM nitrocellulose (Bio-Rad) for 100 min with 400 mA constant. Antibodies were used as follows: Anti-Flag Ms (1:2000; Sigma), anti-FUS H6 Ms (1:1000; Santa Cruz Biotechnology), anti-MeCP2 D4F3 (1:2000; Cell Signaling), and anti-Actin Rb (1:2000; Sigma) were all diluted into 4% nonfat milk (Lab Scientific) and Tris-buffered saline supplemented with 0.5% Tween (TBST). Protein bands were visualized using Millipore ECL kit and CL-X Posure X-ray film (Thermo-Scientific).