Anti phospho p38 antibody

Protein samples were reduced with DTT and loaded onto NuPAGE Bis-Tris gels to detect total and phosphorylated JNK, p-38, and p-65, HSP90A, PKR, and PACT and onto NuPAGE Tris-Acetate gels to detect AHNAK. For immunodetection, gels were transferred onto PVDF membranes. The membranes were blocked with PVDF blocking agent (Toyobo, Osaka, Japan), and then incubated with anti-JNK antibody (#9252; Cell Signaling Technology, Danvers, MA), anti-phospho-JNK antibody (#4668; Cell Signaling Technology), anti-total p-38 antibody (#8690; Cell Signaling Technology), anti-phospho-p-38 antibody (#4511; Cell Signaling Technology), anti-total p65 antibody (#8242; Cell Signaling Technology), anti-phospho-p65 antibody (#3033, Cell Signaling Technology), anti-HSP90α antibody (GTX109753; GeneTex, Irvine, CA), anti-AHNAK antibody (ab178317; Abcam, Cambridge, MA), anti-PKR antibody (ab32506; Abcam), or anti-PACT antibody (ab75749; Abcam). The immunoreactive proteins were identified using a Westernbright Quantum Kit (K-12042-D20; Advansta, Menlo Park, CA).

Purified rhBMP-2, IL-6, and sIL-6R were provided by R&D Systems (Minneapolis, MN, USA). Monensin, dorsomorphin homolog 1 (DMH1), naphthol AS-BI alkaline solution, and phalloidin were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Rabbit anti-Smad1 antibody, rabbit anti-BMPR1A antibody, mouse anti-BMPR2 antibody, rabbit anti-CCAAT enhancer-binding protein-α (C/EBPα) antibody and rabbit anti-peroxisome proliferator-activated receptor gamma (PPARγ) antibody were obtained from Abcam (Cambridge, MA, USA). Anti-phospho-Smad1/5/8 antibody, anti-phospho-p38 antibody, anti-p38 antibody, anti-Runx2 antibody, anti-GAPDH antibody, anti-β-actin antibody, and anti-fade reagent were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-BMPR1B antibody, Alexa Fluor-488-conjugated secondary antibody, and TRIzol reagent were obtained from Invitrogen (Carlsbad, CA, USA). FuGENE®HD transfection reagent was provided by Promega BioSystems (Sunnyvale, CA, USA). Smad1 shRNA plasmids were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Human MSC osteogenic and ADM were purchased from Cyagen Biosciece (Guangzhou, China). Lewis rats were provided by Shanghai Experimental Animal Center China.

RANKL was purchased from Peprotech (London, UK). Alpha-minimum essential media (α-MEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and Dulbecco’s phosphate buffered saline (DPBS) were obtained from Gibco (Gaithersburg, NY, USA). TRAP assay kit was obtained from Sigma Aldrich (Saint Louis, MO, USA). Osteo assay surface multiple well plates were obtained from Corning, Inc. (New York, NY, USA). Anti-c-Fos antibody, anti-TRAF6 antibody and anti-β-actin antibody were obtained from Santa Cruz (CA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA). Anti-MMP-9 antibody and anti-CTK antibody were purchased from Abcam (Cambridge, MA, USA). Anti-total-ERK antibody, anti-phospho ERK antibody, Anti-total-JNK antibody, anti-phospho JNK antibody, Anti-total-p38 antibody and anti-phospho p38 antibody were purchased from Cell signaling (Beverly, MA, USA). Anti-NFATc1 antibody was purchased from BD Pharmingen (San Diego, CA, USA).PCR primers were obtained from Genotech (Daejeon, Korea). All of the chemicals used in the experiments were of analytical grade or complied with the level required for cell culture.

Exponentially growing cells (25 ml) were harvested by centrifugation (3,000 rpm for 1 min), before and after exposure to the indicated stress agent. The supernatants were discarded, and the pellets were snap-frozen in liquid nitrogen. The pellets were thawed and washed in ice-cold lysis buffer (20 mM HEPES [pH 7.3], 350 mM NaCl, 10% glycerol, 0.1% Tween 20) containing protease inhibitors (2 μg/ml pepstatin A, 2 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride [PMSF], 20 μg/ml aprotinin) and phosphatase inhibitors (2 mM sodium orthovanadate, 50 mM sodium fluoride). The cells were resuspended in 200 μl lysis buffer and transferred to a ribolyser tube containing 1 ml chilled glass beads. The samples were disrupted by bead beating (BioSpec) for 2 × 15 s. Lysates were recovered by centrifugation, and 30 µg of extract was subjected to SDS-PAGE on 10% gels. Hog1 was detected by Western blot analysis using an anti-Hog1 antibody (y-215; Santa Cruz Biotechnology). Phosphorylated Hog1 was detected with an anti-phospho-p38 antibody (9211; Cell Signaling Technology) as described previously (43 (link)). Protein loading in some experiments was determined using an anti-tubulin antibody (DSHB, University of Iowa). The experiments were performed three times.

TCA extracts were prepared as described [30 (link)]. Atf1 was detected with a polyclonal anti-Atf1 antibody [31 (link)], phosphorylated Sty1 was detected with an anti-phospho-p38 antibody (Cell Signaling, 9215), and total Sty1 protein was detected with a polyclonal antibody [32 (link)].