Anti phospho prb

Immunoblotting was performed as described previously.^{50 (link)} Briefly, whole-cell extracts or immunopurified proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (6–12%) and transferred to a HyBond ECL nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). The following antibodies were used as primary antibodies: anti-ACOT7 (Abcam, Cambridge, UK), anti-pRb (Cell Signaling, Danvers, MA, USA), anti-phospho-pRb (Cell Signaling), anti-cleaved PARP (Cell Signaling), anti-p53 (Do-7; Leica, Milton Keynes, UK), anti-p21 (Santa Cruz Biotech, Dallas, TX, USA), anti-cyclin D1 (Santa Cruz Biotech), anti-CDK2 (Santa Cruz Biotech), anti-CDK4 (Santa Cruz Biotech), anti-phospho-PKCα/β II(Thr638/641) (Cell Signaling), anti-phospho-PKCδ/θ(Ser643/676) (Cell Signaling), anti-phospho-PKCζ/λ(Thr410/403) (Cell Signaling), and anti-actin (Santa Cruz Biotech).

Paraffin or frozen tumor tissue sections were prepared for collagen sirius red staining, histological analysis, BrdU incorporation assays, TUNEL assays, senescence‐associated‐beta‐galactosidease (SA‐β‐gal) staining and immunostaining according to published protocols 15, 16, 17. The following primary antibodies were used: anti‐TE‐7 (Millipore, Billerica, MA, USA, 1:200), anti‐β‐catenin (Invitrogen, Carlsbad, CA, USA, 15B8, 1:1000), anti‐PDGFR‐α (R&D, Minneapolis, MN, USA, 1:40), anti‐prolyl‐4‐hydroxylase‐ β(P4HB) (Abcam, Cambridge, MA, USA, 1:200), anti‐BrdU (Abcam, BU1/75, 1:25), anti‐Ki67 (Imgenex, Littleton, CO, USA, 1:50), anti‐Cyclin D1 (Cell Signaling, Danvers, MA, USA, 1:50), anti‐E‐cadherin (cell signaling, 24E10, 1:400), anti‐Vimentin (Abcam, 1:200), anti‐N‐cadherin (Abcam, 1:200), anti‐phospho‐pRb (cell signaling, 1:200). The images were taken under a Nikon Eclipse 80i fluorescence microscope.

Cells were lysed as was described previously (6). Protein samples were separated by either 4% or 12%-SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with subsequent transfer onto a 0.45 µm nitrocellulose membrane (BioRad, Tokyo, Japan). Blots were blocked with 5% nonfat dry milk in Tris-buffered saline supplemented with 0.05% Tween 20 for 1 h at room temperature and then incubated overnight at 4 °C with following primary antibodies: anti-phospho-pRb (Ser807/811, 1:1000), anti-cyclin A2 (1:2000), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:1000), anti-geminin (1:1000), anti-phospho-p53 (Ser15, 1:1000), anti-p21Waf1/Cip1 (1:1000), anti-phospho-ATM (Ser1981, 1:1000), anti-cyclin D1 (1:1000, all from Cell Signaling Technology, Danvers, MA, USA), and anti-Emi1 (1:1000, Abcam, USA). Blots were washed, incubated with peroxidase-conjugated goat anti-mouse IG (GAM-HRP, 1:10,000) and goat anti-rabbit IG (GAR-HRP, 1:10,000, both from Cell Signaling Technology, USA) for 1 h at room temperature and developed with ECL (Thermo Scientific, USA). Hyperfilm (CEA) was from Amersham. For densitometric analysis of protein bands ImageJ software (US National Institutes of Health) was used.

GV1001, a human telomerase-derived 16-mer peptide, was provided by GemVax-KAEL (Seongnam, Republic of Korea). The following agents were obtained from commercial sources: vascular endothelial growth factor-A 165 (Merck Millipore, Billerica, MA, USA); anti-phospho-VEGFR-2 (Y1175), anti-phospho-MEK (S217/S221), anti-MEK, anti-phospho-Src (Y416), anti-Src, anti-phospho-p70^S6K (T421/S424), anti-phospho-Akt (S473), anti-phospho-ERK (T202/Y204), anti-phospho-pRb (S780), and anti-phospho-pRb (S807/S811) (Cell Signaling Technology, Beverly, MA, USA); fibroblast growth factor-2 (FGF-2), anti-phosphotyrosine, anti-phospho-FAK(Y397), anti-FAK, anti-β-catenin, and anti-p120-catenin (BD Biosciences, Bedford, MA, USA); anti-vascular endothelial (VE)-cadherin, anti-VEGFR-2, anti-p70^S6K, anti-Akt, anti-ERK, anti-Cdk2, anti-Cdk4, anti-cyclin D, anti-cyclin E, anti-p27^kip1, anti-actin antibodies, and mouse and rabbit IgG-horseradish peroxidase conjugates (Santa Cruz Biotechnology, Santa Cruz, CA, USA); goat anti-mouse IgG-Alexa Fluor 488 conjugate (Thermo Fisher Scientific Co., Waltham, MA, USA).