No complications occurred from the biopsy procedure. Real-time quantitative RT-PCR

Total RNA from rectus abdominis MK1775 muscle was extracted by TRIzol reagent and cDNAs were reverse-transcribed by Revert Aid TM reverse transcriptase. Real-time PCR was carried out using the ABI PRISM 7700 Sequence Detection System (Applied Bio systems) at 50°C for 2 min, 95°C for 10 min, followed by 50 cycles at 95°C for 15 s, and at 60°C for 1 min. The primers for GAPDH (224 bp) were 5′-TGAAGGTCGGAGTCAACGG-3′ (sense) and 5′- CTGGAAGATGGTGATGGGATT-3′ (antisense). The primers for TRAF6 (134 bp) were 5′-GCCTGGGTGACAGAGTGC-3′ selleck inhibitor (sense) and 5′-AATGACTACTTATGGCTCCTTTTC-3′ (antisense). The primers for ubiquitin(165 bp) were 5′-CCCTGGATGTGATGGTGTC-3′ (sense) and 5′-CTCGTTGTCCCTGTTGCTG-3′ (antisense). The expression of GAPDH was used to normalize that

of the target genes. mTOR inhibitor Each assay was done in triplicate, the average was calculate, and the expression level of TRAF6 and ubiquitin was expressed as 2–ΔΔCt, ΔCt = Ct (Target)–Ct (GAPDH). Immunoblotting Cells were lysed in RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.5, 1% NP40, 1% deoxycholate, 0.1% SDS, protease inhibitor cocktail (Roche)). Total proteins were fractionated using the NuPAGE 4–12% Bis-Tris gradient gel (Invitrogen) and transferred onto PVDF membrane. Membranes were blocked with 5% non-fat milk in PBS/Tween-20, and incubated with antibodies against TRAF6 (Santa Cruz), ubiquitin (Santa Cruz), and β-actin (Abcam). Statistical analysis In order to analyze the relationship among the expression of TRAF6 and ubiquitin and nutritional status of

patients (percent weight loss, serum albumin), according to the literature [12], they were divided into two groups(percent weight loss ≥ 10 and <10, serum albumin ≥ 35and <35). All statistical analyses were performed using SPSS16.0 software. Measurement data were analyzed using the Student’s t test, while categorical data were studied using χ2 or Fisher exact tests. Statistical significance was set at P < 0.05. Results The expression of TRAF6 in muscle of control and cancer patients Tumor necrosis factor (α) receptor adaptor protein 6(TRAF6), Astemizole a protein involved in receptor-mediated activation of several signaling pathways, is enhanced in skeletal muscle during atrophy. We assessed the expression of TRAF6 in 29 control muscles and 102 patient muscles. TRAF6 was significantly upregulated in muscle of gastric cancer compared with the control muscles (P < 0.05). TRAF6 was upregulated in 67.65% (69/102) muscle of gastric cancer. Overexpression of TRAF6 in muscles of gastric cancer were associated with TNM stage, level of serum albumin and percent of weight loss (P > 0.05) (Table 2). We also analyze the expression of TRAF6 in 8 muscles of control and cancer patients by western blotting, the results show the expression of TRAF6 in muscle of cancer patients were higher than control (Figure 1).