Anti mtor

RIPA buffer (MilliporeSigma; Merck KGaA) was used to extract total proteins from cultured 22Rv1 or PC-3 cells. Proteins were quantified with a BCA method and separated by 12% SDS-PAGE, and transferred onto PVDF membranes (GEHealthcare). The membranes were blocked in 5% non-fat dry milk diluted with TBST (Tris–HCl 20 mmol/L, NaCl 150 mmol/L, pH 7.5, 0.1% Tween 20) at room temperature for 1 h and washed with TBST for three times. Subsequently, specific primary antibodies (anti-Vimentin, anti-N-cadherin, anti-a-SMA, anti-E-cadherin, anti-Claudin 1, anti-GAPDH, anti-p-Akt, anti-Akt, anti-p-mTOR, anti-mTOR, anti-PTEN, anti-p-Smad2, anti-Smad2, anti-p-Smad3, anti-Smad3, anti-p-Smad2/3, anti-Smad2/3, and anti-laminB, Sigma, USA) were incubated with the membranes at 4 °C overnight. Next, the membranes were washed with TBST for three times and incubated with the secondary antibodies at room temperature for 2 h. Proteins were visualized using chemiluminescence using ECL luminescence reagent (Absin Bioscience Inc., Shanghai, China). The band intensities were quantified using Image-Pro Plus 6.0 (Media Cybernetics, Inc.). GAPDH was used as a loading control for normalization. The primary antibodies and secondary antibodies used in western blotting are included in Table 1.

Whole-cell lysates were prepared from cells collected in lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 1.0% SDS, 2 mM NaF, 2 mM Na₃VO₄, and protease inhibitors (Roche) followed by sonication and centrifugation at 13,000g for 10 minutes. Tumor lysates were prepared by homogenizing tumor tissue in lysis buffer and centrifuging at 13,000g for 10 minutes at 4°C. Proteins were quantified using a BCA assay kit, separated by electrophoresis on SDS PAGE gels, and transferred to PVDF membranes. After one hour in blocking buffer (SuperBlock), membranes were incubated overnight with primary antibodies. The following antibodies were used: anti-p-Akt (Cell Signaling), anti-Akt (Cell Signaling), anti-rpS6 (Abcam), anti-p-rpS6 (Abcam), anti-mTOR (Sigma-Aldrich), anti-p-mTOR (Sigma-Aldrich), anti-ERK (Abcam), anti-p-ERK (Abcam), anti-LC3B-II (Abcam), anti-Atg7 (Santa Cruz Biotechnology), anti-Atg12 (Santa Cruz Biotechnology), anti-Beclin-1 (Abcam), anti-C/EBPα (Abcam), anti-PPARγ (Abcam), anti-FABP4 (Abcam), anti-FAS (Abcam), anti-Tubulin (Abcam), and anti-β-Actin (Santa Cruz Biotechnology). The anti-Tubulin and anti-β-Actin were used as controls. Specific protein bands were imaged using secondary antibody conjugated with horseradish peroxidase and chemiluminescent ECL reagents. Image J was used for quantification.

Cells were collected and lysed. The protein contents were determined. Samples were resolved by SDS-PAGE and electroblotted onto a polyvinylidene difluoride membrane. Afterward, membranes were incubated respectively with an anti-Belcin1(1:1000 dilution, Abcam, USA), anti-LC3(1:1000 dilution, Abcam, USA), anti-phosphorylated PI3K (p-PI3K, 1:1000 dilution, Abcam, USA) and total PI3K (t-PI3K, 1:1000 dilution, Abcam, USA), anti-phosphorylated Akt (p-Akt, 1:1000 dilution, Millipore, USA) and total Akt (t-Akt, 1:1000 dilution, Millipore, USA), anti-mTOR (1:1000 dilution, Millipore, USA) and anti-GAPDH antibodies 1:1000 dilution, Millipore, USA) overnight at 4°C, followed by incubation with the horseradish peroxidase-conjugated anti-rabbit IgG for 1 h at room temperature. The signals were enhanced using a chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, England).