Anti phospho p44 42 erk

Cell lysates (40 μg protein/line) were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The blotted membranes were blocked with 5% skim milk or 5% bovine serum albumin and incubated overnight at 4 °C. Anti-PEAK1 (86 kDa, Abnova, Taipei, Taiwan), anti-EGFR (Tyr1173, 175 kDa), anti-EGFR (175 kDa), anti-KRas (21 kDa), anti-phospho-p44/42 Erk (Thr202/Tyr204, 44/42 kDa) anti-p44/42 Erk (44/42 kDa) (Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (43 kDa) and anti-GAPDH (36 kDa) (Abcam, Cambridge, UK) antibodies were used. The detailed procedures are described in the Supplemental materials and methods.

The C2C12 mesenchymal cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The adenovirus-BMP2 (Adv-BMP2) and adenovirus-β-Gal (Adv-β-Gal) were provided by Dr. Jueren Lou (Institute of Biological Products, Shanghai, China). The Human TNF-α was purchased from PeproTech (300-01A; Rocky Hill, NJ). Real-time PCR was done via the ABI7900HT system using SYBR1Premix Ex Taq^TM (DRR041A; Takara, Dalian, China). The anti-SATB2 (1:1000; SATBA4B10), anti-Lamin B (1:500; ab151735) and anti-TNF-α (1:600; ab34674) antibodies were obtained from Abcam (Cambridge, MA). The anti-GAPDH antibody (1:10000; sc-32233) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-p44/42 ERK (1:1000; #4370) and anti-total-p44/42 ERK (1:1000; #4695), anti-phospho-p38 (1:1000; #4631) and anti-total-p38 (1:1000; #8690), and anti-phospho-JNK (1:1000; #4668) and anti-total-JNK (1:1000; #9252) antibodies, the anti-P65 (1:1000; #8442) and anti-beta-actin antibodies (1:10000; #3700) were purchased from Cell Signaling Technology (Danvers, MA).

Total cell lysates were extracted using CytoBuster™ Protein Extraction Reagent (Novagen). Equal amounts of protein were separated on 10 to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels at 300 mA for 20 min. They were then transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Billerica, MA, USA) using a trans-blot SD Semi-Dry Transfer Cell (Bio-Rad, Hercules, CA, USA). After transfer, the membranes were incubated in Tris buffered saline (TBS), 5% dry milk, and 0.2% Tween 20 for 1 h. They were then further incubated in TBS and 0.2% Tween 20 with specific antibodies at 4 °C overnight. After washing, a horseradish peroxidase (HRP)-labeled secondary antibody was applied and the membranes were incubated for 2 h. Immunoblot detection was performed using Immobilon Western HRP Substrate (Millipore Sigma). The following antibodies from Cell Signaling Technology were used: anti-protein kinase B (PBK/Akt), anti-phospho-Akt (Ser473), anti-c-jun N-terminal kinase (JNK), anti-phospho-JNK, anti-p38, anti-phospho-p38, anti-p44/42 extracellular signal-related kinase (ERK), anti-phospho-p44/42 ERK, and anti-GAPDH.