Anti pt202 py204 erk1 2

The MG-63 osteosarcoma cell line was provided by RIKEN BioResource Center through the National BioResource Project of the Ministry of Education, Culture, Sport, Science and Technology of Japan (Tsukuba, Ibaraki, Japan). Anti-AKT, anti-pS473 AKT, anti-p38, anti-pT180/pY182 p38, and anti-pT202/pY204 ERK1/2 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-ERK2 antibody was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The following pharmacological inhibitors were used: BAPTA-AM (membrane-permeable calcium chelator) from Focus Biomolecules (Plymouth Meeting, PA, USA), LY294002 (PI3-kinase inhibitor) from Cayman Chemical (Ann Arbor, MI, USA), dabrafenib (RAF-1 and B-RAF inhibitor) from LKT Laboratories (Saint Paul, MN, USA), and Y-27632 (Rho kinase inhibitor) from Nacalai Tesque (Kyoto, Japan). Porcine type I collagen (Cellmatrix Type I-C) was purchased from Nitta Gelatin (Osaka, Japan). Other reagents were purchased from Nacalai Tesque unless specified otherwise.

After stimulation with rmIL-33 and/or rmSCF (both 50 ng/mL) (Peprotec), BMMCs were lysed in lysis buffer (20 mM HEPES, pH 7.5; 10 mM EGTA, 40 mM β−glycerophosphate, 2.5 mM MgCl₂, 2 mM orthovanadate, 1 mM dithiothreitol, 20 µg/mL aprotinin, and 20 µg/mL leupeptin supplemented with 1% Triton). The protein concentration was determined (BCA−kit; Pierce), and the samples were boiled (with 6 × Laemmli buffer). The lysates were separated with 10% sodium dodecyl sulfate (SDS) −Laemmli gels and afterwards transferred onto nitrocellulose membranes (biostep) by Western blotting. The membranes were blocked (with dry milk) and incubated with anti-pT180/pY182−p38, anti-p38, anti-pS177/181−IKK2, anti-p184/187−pTAK1, anti-pT202/pY204−ERK1/2, anti-pS32−IκBα, anti-IκBα, anti-pS536−p65, anti-p65, HSP90, anti-pST183/Y185−JNK1/2, anti-JNK1/2, anti-pS727−STAT3, anti-STAT3, anti-pS235/236−S6, anti-S6, and anti-tubulin (all from Cell Signaling except anti-IKK1/2, anti-TAK1, and anti-ERK1/2 (Santa Cruz, CA, USA)). The membranes were washed with TBS/0.1% Tween and incubated with HRP-conjugated secondary anti-rabbit Ig, anti-mouse Ig, or anti-goat Ig (SeraCare). We used ECL reagent (Pierce) for detection.