P eif4ebp1

The protein expression and phosphorylation analysis was performed as described,^66,69 (link) with minor modifications. The primary antibodies used were all purchased from Cell Signaling Technology: SQSTM1/p62 (5114), LC3 (12741), pULK1 (S555; 5869), pRPTOR/RPTOR (Ser792) (2083), p-PRKAA/AMPKɑ (Thr172; 2535), pAKT (Ser473; 4058), p-MTOR (Ser2448; 2971), pRPS6KB1/p70S6K1 (Thr389, Ser371; 9234, 9208), p-EIF4EBP1 (Thr37/Thr46; 2855), pGSK3B (Ser9; 9327), γ-H2AFX/H2AX (Ser139; 9718), pCDC2 (Tyr15; 4539), CCND1/Cyclin D1 (2926), HIST1H3A/C (Ser10; 9701), CDKN1A/p21 (2947), and p-MAPK14/p38 (Thr180/Tyr182; 4511).

Total protein was extracted from skeletal muscle by the protein extraction Kit (KeyGEN). Protein concentration was determined with BCA Protein Assay Kit (Takara), and an equal amount of protein was separated on SDS-polyacrylamide gel electrophoresis. After transferring to a polyvinylidene fluoride (PVDF) membrane (Millipore), the primary antibody and the corresponding secondary antibody were used to detect protein of interest. The antibody used in this study were as follows: Atrogin-1 (Abclonal); MuRF-1 (Abclonal); LC3 (Abclonal); P62 (Abclonal); Ndufb2 (Abclonal); Clusterin (Abclonal); IGF1 (Abclonal); PI3K(p85α) (Abclonal); P-mTOR (Abclonal); mTOR (Abclonal); P-P70S6K (Abclonal); P70S6K (Abclonal); P-FOXO3A (Abclonal); FOXO3A (Abclonal); P-EIF4EBP1 (Abclonal); EIF4EBP1 (Abclonal); P-AKT (Cell Signaling Technology); AKT (Proteintech); and GAPDH (Bioworld Technology). The protein was visualized using High-sig ECL Western Blotting Substrate (Tanon) and imaged using the Tanon-5200S Chemiluminescent Imaging System (Tanon).

The sections were fixed in 4% paraformaldehyde, embedded in paraffin wax, cut into 5 μm sections, de-waxed through xylene, dehydrated through graded alcohols and were prepared for hematoxylin–eosin staining (HE) or immunohistochemical analysis. Antigen retrieval was performed using citrate buffer (pH 6.0) and high-power steam. Tissue sections were then incubated overnight with the primary antibody solution (diluted according to manufacturer’s recommendation) at 4°C. Primary antibodies include mouse anti-human CD31 (Ab24590, Abcam, a typical marker for vascular endothelial cells), alpha-smooth muscle actin (α-SMA, BM0002, BOSTER), rabbit-anti-human mTORC1 (Ab115330, Abcam), p70S6 (Ab32359, Abcam), eIF4EBP1 (Ab2606, Abcam), p-p70S6 (Thr 389, #9234, Cell Signaling Technology), and p-eIF4EBP1 (Thr70, #9455, Cell Signaling Technology) antibodies. The sections were then incubated with secondary antibody for 2 h and DAB Horseradish Peroxidase Color Development Kit (DAB, Dako Code K5007, Agilent Technologies, Santa Clara, CA) and were performed at room temperature. The sections were observed under a light microscope.