Anti phospho 4ebp1

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).

Lung specimens were fixed in 10% formaldehyde, embedded in paraffin, and stained with haematoxylin-eosin (HE) and Elastica-Masson (EMa). Specimens from patients 1 and 2 were examined immunohistochemically using the following commercially available antibodies: anti-p53 (DO-7, 1/200 dilution; Leica Biosystems, Nussloch, Eisfeld, Germany), anti-CEA (II-7, 1/50 dilution; Dako, Santa Clara, CA, USA), anti-Ki-67 (MIB1, 1/200 dilution; Dako), anti-melanosome (HMB45, 1/100 dilution; Dako), anti-phospho-AKT (#3787, 1/25 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-p70S6K (#9206, 1/50 dilution; Cell Signaling Technology), and anti-phospho-4E-BP1 (#2855, 1/25 dilution; Cell Signaling Technology) antibodies.

We used the following antibodies: anti-Akt (No. 9272), anti-phospho-Akt (Ser473; No. 9271), anti-S6K (No. 9202), anti-phospho-S6K (Thr389; No. 9205), anti-S6 (No. 2217), anti-phospho-S6 (Ser235/236; No. 4858), anti-4EBP1 (No. 9644), anti-phospho-4EBP1 (Thr37/46; No. 2855), anti-GSK3β (No. 12456), anti-phospho-GSK3β (Ser9; No. 5558), anti-Ampk (No. 2532), anti-phospho-Ampk (Ser9; No. 2535), anti-mouse IgG (No. 7076), and anti-rabbit IgG (No. 7074) purchased from Cell Signaling Technology (Massachusetts, United States of America). Anti-α-Tubulin (12G10) was purchased from DSHB (Iowa, United States of America). Anti- Laminin α-2 (SC-59854) was purchased from Santa Cruz Biotechnology (Texas, United States of America).