Rabbit anti p27

BCA (purity, 95.8%) was purchased from the Institute for Korea Traditional Medical Industry (Daegu, Korea) and dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). A BCA stock solution of 40 mM was stored at -80°C. Mouse anti-β-actin (1 : 5000 dilution), rabbit anti-p-AKT (1 : 1000 dilution), rabbit anti-AKT (1 : 1000 dilution), rabbit anti-p-p53 (Ser15) (1 : 1000 dilution), rabbit anti-p-p53 (Ser20) (1 : 1000 dilution), rabbit anti-p-p53 (Ser46) (1 : 1000 dilution), and rabbit anti-MDM2 (1 : 1000 dilution) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-p21 (1 : 1000 dilution), rabbit anti-p27 (1 : 1000 dilution), rabbit anti-p53 (1 : 1000 dilution), rabbit anti-FOXO3 (1 : 1000 dilution), rabbit anti-Bcl-2 (1 : 1000 dilution), rabbit anti-Bcl-xL (1 : 1000 dilution), rabbit anti-Bax (1 : 1000 dilution), rabbit anti-cleaved caspase-3 (1 : 1000 dilution), rabbit anti-caspase-3 (1 : 1000 dilution), rabbit anti-cleaved caspase-7 (1 : 1000 dilution), rabbit anti-caspase-7 (1 : 1000 dilution), rabbit anti-cleaved caspase-9 (1 : 1000 dilution), rabbit anti-caspase-9 (1 : 1000 dilution), rabbit anti-cleaved PARP (1 : 1000 dilution), and rabbit anti-PARP (1 : 1000 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA).

The following primary antibodies were employed for Western blotting: mouse anti-β-tubulin (1:1000, cod. T5201; Sigma-Aldrich Corp.), mouse anti-β-actin (1:2000, cod. A5441; Sigma-Aldrich Corp.), rabbit anti-GAPDH (1:1000, cod. G9545, Sigma-Aldrich Corp.), mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem, St. Louis, MO, USA), mouse anti-histone H3 (1:500, cod. 61475; Active Motif, Carlsbad, CA, USA). The secondary antibodies used for Western blot analysis were the following: horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000, cod. 170-6516; Bio-Rad, Hercules, CA, USA) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, cod. 170-6515: Bio-Rad, Hercules, CA, USA). The primary antibodies employed for immunofluorescence staining are listed below: mouse anti-cathepsin D (1:100, cod. IM03; Calbiochem), rabbit anti-cathepsin D (1:100; EMD Biosciences, Calbiochem, San Diego, CA, USA), rabbit anti-p27 (1:100, cod. 2552; Cell Signaling, Danvers, MA, USA), rabbit anti-Ki-67 (1:100, cod. HPA001164; Sigma-Aldrich), mouse anti-E-cadherin (1:50, cod. 14472S; Cell Signaling) and rabbit anti-N-cadherin (1:50, cod. 4061S; Cell Signaling). The secondary antibodies were goat-anti rabbit IgG Alexa Fluor Plus 488 (1:1000, cod. A32731; Invitrogen, Waltham, MA, USA) and goat-anti mouse IgG Alexa Fluor Plus 555 (1:1000, cod. A32727; Invitrogen).

AGS cells were infected with 5 MOI GPX7 expressing and control virus for 48 hours. Then the cells were seeded onto an 8-chamber culture slide. The second day, the cells were fixed with 4% paraformaldehyde in PBS at room temperature for 45 minutes, followed by permeabilization with 0.5% Triton × −100 in PBS for 2 minutes on ice. After blocking, cells were incubated with primary antibodies (rabbit anti-p27 and mouse anti-Cyclin D1, 1:200 respectively, Cell Signaling, Danvers, MA USA) overnight at 4°C, and protected from the light. Cells were washed twice with PBS and incubated with anti-mouse or anti-rabbit secondary antibody labelled with Fluor-488 or Fluor-586 for 1 hour at room temperature, protected from light. After washing, cells were covered with Vectashield mounting medium with DAPI (Vector Laboratories). Cyclin D1 or p27-positive cells were counted (> 400 cells) using ImageJ software. The percentage of Cyclin D1 or p27-positive cells versus total number of cells was calculated and statistically analyzed using Prism software.

Primary antibodies: mouse anti-PARP-1, rabbit anti-phospho-Ser473 Akt, rabbit anti-phospho-Thr308 Akt, rabbit anti-Akt, rabbit anti-phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti-phospho-MEK1/2, mouse anti-MEK1/2, mouse anti-p21waf1, rabbit anti-p27, were from Cell Signaling Technologies (Boston, MA, USA). Mouse anti-human GAPDH and rabbit anti-cyclin A were from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies: horseradish peroxidase (HRP)—conjugated goat anti-rabbit and goat anti-mouse IgG (H + L) antibodies were from Jackson (Baltimore Pike West Grove, PA, USA). EZ-ECL enhanced chemiluminescence detection kit, RPMI1640 medium, L-glutamine, fetal bovine serum, trypsin, antibiotics and phosphate buffered saline (PBS) were from Biological Industries (Beit-Ha-Emek, Israel). Propidium iodide, phosphatase inhibitor cocktails 2 and 3, sulphorodamine B (SRB), trichloroacetic acid and acetic acid were from Sigma Aldrich (St. Louis, MO, USA). ZSTK474, Selumetinib and AEW-541 were from Selleckchem (Houston, TX, USA). Complete mini protease inhibitor cocktail, RNAse A and Triton X-100 were from Roche Diagnostics Gmbl (Mannheim, Germany).

The following antibodies were used: mouse anti-MiTF (Abcam), mouse anti-FANCD2 (Santa Cruz), rabbit anti-FANCD2 (Abcam), rabbit anti-FANCA (Bethyl), mouse anti-Flag (Sigma), rabbit anti-p53 serine 15 (Cell Signaling Technology), mouse anti-p53 (Santa Cruz), rabbit anti p27 (Cell Signaling), rabbit anti-p21 (Santa Cruz), mouse anti-Cyclin-A (Abcam), rabbit anti-53BP1 (Abcam), mouse anti-gH2AX (Millipore), mouse anti-alpha-tubulin (Abcam), mouse anti-vinculin (Abcam), and goat anti-actin (Abcam).