Anti phospho p70 s6 kinase

The peripheral parts of muscle specimens were immediately frozen in liquid nitrogen and stored at −80°C for biochemical studies. Western blotting was performed as previously described (Fujimura and Usuki, 2015 (link), 2017 (link)). Briefly, the samples were sonicated for 5 s in tissue lysis buffer (T-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, USA) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, St Louis, USA). The samples were centrifuged (14,000 g for 1 h), and the supernatants were collected. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 μg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were then subjected to the following antibody probes: anti-MGF (Millipore, Billerica, USA); anti-IGF-I (Santa Cruz Biotechnology, CA, USA); anti-YAP1 (Novus Biologicals, Centennial, CO, USA); anti-phospho-YAP1 (Abcam, Cambridge, UK); anti-PAX7 (Cytoskeleton Incorporated, Denver, USA); anti-phospho-ERK, anti-ERK, anti-AKT, anti-phospho-AKT, anti-phospho-4EBP1, and anti-phospho-p70 S6 kinase (Cell Signaling Technologies); and anti-β-actin (Sigma-Aldrich).

Dental pulp of the mandibular first molars was dissected from Ror2^fl/fl control mice and Osr2-Cre;Ror2^fl/fl mice at P3.5. For kidney capsule transplantation, dental pulp of transplanted molars was dissected from the kidney capsule 2 days post-surgery. For western blotting, tissues were lysed in lysis buffer [50 mMTris-HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 0.1% NP-40, 10% glycerol and protease inhibitor cocktail] and protein fractions were isolated. Proteins were separated on 4-15% protein gels (Bio-Rad) and transferred to a 0.2 µm PVDF membrane. Membranes were blocked in 5% BSA for 1 h at room temperature and then incubated with primary antibody overnight at 4°C, followed by 1 h incubation with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibody. Immunoreactive protein was detected using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, A10044).
The primary antibodies used for western blotting were as follows: anti-GAPDH (1:1000, Cell Signaling Technology, 5174s); anti-Ror2 (1:1000, Cell Signaling Technology, 886395); anti-Cdc42 (1:1000, Abcam, ab64533); anti-phospho-p70S6 kinase (1:1000, Cell Signaling Technology, 9205S); anti-p70S6K (1:1000, Cell Signaling Technology, 2708); anti-mTOR (1:1000, Cell Signaling Technology, 2972s); and anti-phospho-mTOR (1:1000, Invitrogen, 44-1125G).

All three GIST cell lines (GIST882, GIST48, and GIST430) were kindly provided by Dr. JA Fletcher. The mutation status of the KIT gene in these cell lines has been previously described [43 (link)]. GIST882 is an IM-sensitive cell line with a homozygous missense mutation in exon 13 of KIT (K642E) [44 (link)]. The GIST430 line, which harbors a primary, heterozygous, inframe deletion in exon 11 and a secondary, heterozygous, missense mutation in exon 13 in KIT, and the GIST48 line, which harbors a primary, heterozygous, missense mutation in exon 11 and a secondary, heterozygous, missense mutation in exon 17 in KIT, are both relatively IM-resistant [43 (link)]. MLN8237, an AURKA-selective inhibitor, was purchased from Selleck Chemicals. It was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM. Nocodazole was purchased from Sigma-Aldrich (CAS Number 31430-18-9). The following antibodies were used for immunoblotting: anti-Aurora A/AIK (#3092; 1:1000), anti-phospho-Aurora A (Thr288) (#3079; 1:500), anti-phospho-HistoneH3 (Ser10) (pHisH3) (#9701; 1:1000), anti-p53 (#2524; 1:1000), and anti-phospho-p70 S6 Kinase (#9205; 1:500), all from Cell Signaling Technology; anti-p21 (sc-817; 1:1000) and anti-DEC1 (sc-101023; 1:500), both from Santa Cruz Biotechnology; and anti-actin (ABS 24-100; 1:50000).