Rabbit anti p44 42

For Western blots, the protein samples were separated by SDS-PAGE on 12% gels then transferred to nitrocellulose membranes. After blocking the nonspecific binding sites with 3% BSA in TBST (20 mM Tris pH 7.4, 0.1% Tween 20, 150 mM NaCl), the membranes were incubated with rabbit anti-phospho-p44/42 (1:1000 dilution; Cell Signaling, Danvers, MA, USA), or rabbit anti-p44/42 (1:1000 dilution; Cell Signaling) antibodies. The proteins were detected with horseradish peroxidase-conjugated secondary antibodies using the West Save Gold western blot detection kit (Ab Frontier, Seoul, Korea). The immunoreactive bands were visualized using a Chemiluminescence imaging system (Syngene, Cambridge, United Kingdom).

Samples were lysed in 50 μL of 1× RIPA buffer, and protein concentration was determined using the Bio-Rad DC Protein Assay. Samples containing equal amounts of protein were prepared using 5× lane marker-reducing sample buffer. The prepared samples were separated using SDS-PAGE using a 10% polyacrylamide gel, and then transferred to a nitrocellulose membrane at 230 mA for 90 min at 4 °C. Primary antibodies including rabbit anti-phospho-p44/42 (1:1000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-p44/42 (1:1000; Cell Signaling Technology), and rabbit anti-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were incubated with the membrane overnight at 4 °C. The secondary antibody was horseradish-peroxidase-conjugated anti-rabbit IgG (1:5000; Cell Signaling Technology), which was incubated for 1 h at around 28 °C. Proteins were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West FemtoMaximum Sensitivity Substrate (Thermo Fisher Scientific). The membranes were stripped with Restore Western Blot Stripping Buffer for probing with different antibodies.

BMMs were activated as indicated, and the protein lysates were subjected to Western blots. The primary antibodies used in this study were as follows: rabbit anti-Notch1 (1:2000) (Santa Cruz Biotechnology, USA), rabbit anti-cleaved Notch1 (Val1744) (1:1000), rabbit anti-phospho-p38 (1:2000), rabbit anti p38 (1:2000), rabbit anti-phospho-p44-42 (1:4000), rabbit anti p44-42 (1:4000), rabbit anti-phospho-SAPK-JNK (1:2000), rabbit anti-SAPK-JNK (1:2000), rabbit anti-phospho-AKT (1:2000), rabbit anti-AKT (1:2000) and rabbit anti-RBPJSHU (1:1000) (all from Cell Signaling Technology, USA), mouse anti β-actin (1:1000) (Chemicon-Millipore, USA) and rabbit anti-GAPDH (1:4000) (Santa Cruz Biotechnology, USA). The secondary reagents conjugated with horse-radish peroxidase (HRP) were as follows: donkey anti-rabbit IgG (1:2000–1:4000) and sheep anti-mouse IgG (1:5000) (Amersham Biosciences, UK). The signals were detected by chemiluminescence on X-ray films.