Phosphorylated erk

Equal amounts of total protein were separated by SDS-PAGE and then transferred to nitrocellulose membranes by semi-dry blotting as previously described [23 (link),24 (link)]. After blocking the membranes with 5% non-fat dry milk, they were probed with antibodies to either phosphorylated p38, JNK, Akt (Cell Signaling, Beverly, MA), phosphorylated ERK (Santa Cruz Biotechnology, Santa Cruz, CA), phosphorylated IκB (New England BioLabs, Beverly, MA), anti-MyD88, ATG16L1, Beclin-1, Atg5, rabbit anti-LC3 (Cell Signaling, Beverly, MA), or anti-NOD1 and NOD2 (Cayman Chemical, Ann Arbor, MI), and then developed with horseradish peroxidase-conjugated second antibodies (Zymed Laboratories, San Francisco, CA) and enhanced chemiluminescence (Pierce Chemical Co., Rockford, IL). Appropriate exposures to X-ray film were made, and the filters then stripped and re-probed with antibodies to GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA) as appropriate.

Berberine sulfate (Purity > 98%) was purchased from Mansite (Chengdu, China), and dissolved in Dimethyl sulfoxide (DMSO). α Modification of Minimal Essential Medium (α-MEM) and fetal bovine serum (FBS) was purchased from TRACE (Sydney, Australia). Recombinant GST-rRANKL protein was expressed and purified as previously described [26 (link)] and recombinant macrophage colony stimulating factor (M-CSF) was obtained from R&D Systems (Minnneapolis, MN, USA). MTS reagent and luciferase analysis reagents were obtained from Promega (Sydney, Australia). Antibodies against NFATc1, IκBα, ERK, phosphorylated ERK, and β-actin were obtained from Santa Cruz Biotechnology (Dallas, CA, USA). Tartrate resistant acid phosphatase (TRAcP) enzymatic activity was detected using the Leukocyte acid phosphatase staining kit (Sigma, St. Louis, MO, USA).

Proteins were extracted from cultured fibroblasts obtained from patients with SCI and healthy subjects and were dissolved in RIPA buffer, boiled for 5 min, and loaded onto 4–12% Bis-Tris gels. Then, separated proteins were blotted onto polyvinylidene difluoride membranes (Invitrogen) with 20% (v/v) methanol in NuPage Transfer Buffer (Invitrogen) at 15 V for 4 h at 4 °C. The membranes were blocked for 1 h in tris-buffered saline containing 0.01% Tween 20 with 5% skim milk (Difco; BD Biosciences, Oxford, UK), then washed three times with tris-buffered saline containing 0.01% Tween 20 for 10 min. The blots were incubated overnight at 4 °C with the following primary antibodies specific to the target proteins: PLK1, CCNB2, CDC20 (1:1000; Abcam, Cambridge, England), CCNB1, BUB1, ERK, phosphorylated ERK, AKT, phosphorylated AKT, and GAPDH (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The next day, the blots were washed three times with TBST and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies (1:4000; Santa Cruz, CA, USA) at room temperature. The blots were washed three times with TBST, then visualized with an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Little Chalfont, UK).

RLE-6TN cells were lysed using RIPA buffer containing 1 mM PMSF, 1μg/mL aprotinin, 1μg/mL leupeptin, 1 mM Na₃VO₄ and 1 mM NaF, and stored in aliquots at -80°C until further analysis. The lysate (20 μg) was mixed with an equal volume of sample buffer, denatured by boiling, and then separated on 10-15% polyacrylamide mini-gel. The proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL), blocked with 5% milk and incubated overnight with E-cadherin (Abcam, Cambridge, MA), Snail (Abcam), p-GSK3β (s9) (Cell Signaling Technology, Danvers, MA), GSK3β (Cell Signaling Technology), α-SMA (Abcam), phosphorylated ERK (Santa Cruz Biotechnology), ERK (Santa Cruz Biotechnology), p-p38, p-JNK (Cell Signaling Technology) and β-actin antibodies (Sigma, St. Louis, MO). The blots were then incubated with anti-mouse or anti-rabbit IgG horseradish peroxidase conjugated antibodies (GE Healthcare, Piscataway, NJ) for 1 h at room temperature. Finally, the signal was detected using Amersham ECL plus (GE Healthcare).

After 1 h exposure with ASD and KRG or Rg3, BEAS-2B cells were harvested and lysed in an ice-cold lysis buffer (Thermo Scientific, Rockford, IL USA). Collected whole cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate protein and transferred onto a nitrocellulose membrane (Bio-Rad, Berkeley, CA, USA). The membranes were blocked with 5% skim milk solution and incubated with the following antibodies against nuclear factor kappa B (NF-κB): phosphorylated NF-κB, C-Jun, phosphorylated C-Jun, p38, phosphorylated p38, ERK, phosphorylated ERK, JNK, phosphorylated JNK, and GAPDH (Santa Cruz Biotechnology). After 1 h incubation, membranes were washed with Tris-buffered saline with 0.1% Tween 20 and then treated with peroxidase-conjugated anti-rabbit immunoglobulin G (Santa Cruz Biotechnology). Bands were visualized using horseradish peroxidase conjugated secondary antibodies and an enhanced chemiluminescence system (Pierce, Rockford, IL, USA). Band densities were measured using the multi Gauge v.2.02 software (Fujifilm, Tokyo, Japan) and expressed as a percentage of treated versus untreated cells.