Anti p akt

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.

Immunocytochemistry was performed and modified according to Iida’s study. Briefly, SH-SY5Y cells were washed with PBS three times, fixed with PBS containing 4% (wt/vol) paraformaldehyde for 15 min, and then permeabilized with 0.5% (wt/vol) Triton X-100 in PBS for 20 min. Immunocytostaining was performed with anti-Akt (1:200), anti-P-Akt (1:200), anti-PI3K (1:200), anti-P-PI3K (1:200), anti-BAD (1:200), anti-Bax (1:100), anti-Bcl-2 (1:100), anti-Cytc (1:200), anti-GSK3β (1:200), anti-p53 (1:100), anti-NGF (1:200), and anti-TrkA (1:200) antibodies (Sigma). After the nonspecific reaction was blocked with PBS containing 10% (wt/vol) bovine serum albumin (BSA), the cells were incubated with the primary antibody in PBS overnight, washed with PBST, and incubated with the second antibody (1:200) in PBST for 1 h. After the samples were washed with PBS three times, they were embedded in DAPI for 5 min and then washed with PBST four times. The images were obtained using an Olympus microscope (Shanghai, China). The mean fluorescence intensity was calculated by Image-Pro software (Meyer, TX, USA).

B cells were lysed in protein lysis buffer containing phosphatase inhibitor (Therm Pierce). Lysates were centrifuged for 15 min at 14,000 × g at 4°C, and protein concentration was determined by Bradford protein assay (Bio-Rad, CA, USA). Proteins were separated by SDS-PAGE using 8% polyacrylamide gels. Proteins were then transferred onto PVDF membranes (Millipore, MA, USA). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) buffer and immunoblotted with primary antibodies including anti-β-actin (Sigma, MA, USA), anti-EBF1 (Sigma, MA, USA), anti-AKT (Sigma, MA, USA), anti-pAKT (Sigma, MA, USA), and anti-P53 (Sigma, MA, USA). Band intensity was quantified using Quantity One software (Bio-Rad, CA, USA).

Cells were mixed with RIPA buffer (Yeasen Biotech, Shanghai, China) supplemented with protease and phosphatase inhibitors and then homogenized in an ice bath at 4 °C for 30 min. After centrifugation at 10,000×g at 4 °C for 20 min, the supernatant was obtained, and the protein concentration was measured using a bicinchoninic acid reagent kit (Yeasen Biotech). Equal amounts of protein (30 μg) were subjected to 10% SDS-PAGE and then transferred onto a nitrocellulose membrane. Standard Western blotting was conducted as previously described [11 (link)] using the primary antibodies anti-TIPE2 (1:1,500 dilution; Abcam, Cambridge, MA, USA), anti-calponin (1:2,500 dilution; Abcam), anti-SM22α (1:2,000 dilution; Abcam), anti-p-PI3K (1:1000 dilution; Cell Signaling Technology, Boston, MA, USA), anti-PI3K (1:1500 dilution; Cell Signaling Technology), anti-p-Akt (1:800 dilution; Sigma-Aldrich), anti-Akt (1: 2500 dilution; Sigma-Aldrich), anti-β-actin (1:1500 dilution; Cell Signaling Technology), and secondary antibody (1:1000 dilution; Cell Signaling Technology). Quantitative protein analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

3-(4,5-dimethyl-2-thiazolyl)-2,5-dipheniyl-2H tetrazolium bromide (MTT), ranolazine (Rn) (10⁻⁶ M), and insulin (Ins) (10⁻⁸ M) were obtained from Sigma-Aldrich biotechnology. anti-MnSOD (SAB2702309) (1:250), anti-NF-κB (MAB3026) (1:250), anti-IκB (ZRB1144) (1:250), anti-PPAR-γ (SAB4502262) (1:300), anti-COX-2 (SAB4503384) (1:500), anti-Cu/Zn-SOD (MABC684) (1:500), anti-AKT (SAB3701427) (1:500), anti-p-AKT (Ser473) (05-1003) (1:500), anti-eNOS (SAB5700744) (1:250), anti-phospho-eNOS (Ser1177) (07-428-I), anti-ERK1/2 (M5670) (1:500), anti-p-ERK1/2 (pThr202/Tyr204) (SAB4301578) (1:500), and anti-β-tubulin (T8328) (1:3000) antibodies (Sigma Aldrich, Madrid, Spain) were used. All other reagents were of analytical- or culture-grade purity.

Protein samples from hair follicles or cultured DP cells were extracted using RIPA buffer (Beyotime, Nanjing, JS, China). Equal amounts of protein were loaded in each lane, resolved on 10% sodium dodecyl sulfate poly-acrylamide gradient gels and transferred onto a 0.45-μm NC membrane (Milipore). The membrane was blocked with 5% bovine serum albumin and incubated with anti-p-Smd2, anti-Smd2, anti-p-Akt, anti-Akt, anti-β-catenin (1:1000; Milipore) or Laminin B1 (polyclonal antibody, 1:1000; Sigma) overnight at 4°C. Followed, then by anti-rabbit or anti-mouse IgG peroxidase conjugate. Immunoreactive bands were visualized by the Immobilon Western hemiluminescent HRP Substrate (Milipore).

Western blotting analysis was performed according to standard methods as previously described.^{12 (link)} The following primary antibodies were used: anti-Sam68 (sc-333, dilution, 1:500; Santa Cruz Biotechnology, Delaware Ave Santa Cruz, CA), anti-beta-catenin, anticyclin D1, anti-p21^cip1, anti-p27^KIP1, anti-p-Rb, antitotal-Rb, anti-p-AKT, antitotal-AKT, anti-c-Myc (1:1000, Millipore, Billerica, MA), anti-P-84, anti-LEF-1 (1:500, Abcam, Cambridge, MA), anti-α-tubulin, anti-TCF-4, anti-LEF1, and anti-GAPDH (1:1000, Sigma, Saint Louis, MO). Nuclear extracts were prepared using the Nuclear Extraction Kit (Active Motif), according to the manufacturer's instructions.