Anti cyclin a2

The following antibodies were obtained commercially: anti-acetylated-tubulin (T7451), anti-γ-tubulin (T5326), and anti-α-tubulin (T9026) (Sigma, St. Louis, MO, USA); anti-LC3A/B (D3U4C) XP (#12741), anti-cyclin A2 (#4656), anti-cyclin E1 (HE12; #4129), anti-CDK2 (#2546), and anti-CDK2 phospho-Thr160 (#2561) were purchased from Cell Signaling (Beverly, MA, USA); anti-fetuin-A (ab137125), and anti-Beclin 1 antibody [EPR19662] (ab207612) were purchased from Abcam (Cambridge, UK); anti-actin (AC-15) (GTX26276) was purchased from Genetex (Irvine, CA, USA); anti-IFT88 (13967-1-AP) was purchased from Proteintech (Chicago, IL, USA); and anti-CEP164 (NBP1-81445) was purchased from Novus (Littleton, CO, USA).

After treatment, the liquid supernatant was taken out and the cells were washed thrice in the surface of 6-well plates. Then, cell lysates were prepared and the concentration of protein was quantified by using BCA protein assay kit (ThermoFisher, USA). Equal amount of proteins was subjected to 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked and probed with specific antibodies (dilution ratio: 1:3000, anti-cyclin A2, anti-cyclin E1, anti-cyclin D2, anti-p-cdc2, anti-CDK2, anti-CDK4, anti-Bax, anti-Bcl-xl, anti-p62, anti-Atg-5, anti-Beclin-1, anti-LC3A/B, and anti-β-actin, Cell Signaling Technology, Danvers, MA, USA) followed by exposure to a horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit antibody and secondary antibodies (dilution ratio: 1:5000, Cell Signaling Technology, Danvers, MA, USA). The immunocomplexes were visualized using a horseradish peroxidase-conjugated antibody, followed by a chemiluminescence reagent (Millipore, Bedford, MA, USA) and detected on photographic film.

Cells were lysed using a lysis buffer (50 mM HEPES, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mM Na2P4O7, 1 mM Na3VO4, 5 ug/mL aprotinin, 5 ug/mL leupeptin, 1 mM PMSF and protease inhibitor cocktail). Lysates containing equal amounts of proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The blots were blocked with a 5% skim milk solution and incubated with the following antibodies: anti-H2A.Z.1, anti-p21, anti-PARP, anti-caspase-9, anti-caspase-3, anti-cleaved caspase-3, anti-p27, anti-Cyclin D1, anti-Cyclin A2, anti-CDK2, anti-p-pRb, anti-vimentin, and anti-slug (Cell Signaling Technology, Danvers, MA), anti-GAPDH, and anti-Fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-E-cadherin, and anti-N-cadherin (BD Transduction, San Jose, CA), and anti-Snail (Abcam, Cambridge, MA). The Immobilon^™ western blot detection system (Millipore, Billerica, MA) was used to detect bound antibodies. The intensities of the western blot bands were quantified using LAS-3000 (Fuji Photo Film Co., Japan).

Sections were deparaffinized, rehydrated, subjected to antigen retrieval and blocked. Then the tissues were incubated with the following antibodies: anti-SOSTDC1, anti-cyclin E2 (Abcam, Cambridge, MA), anti-cyclin A2 (Cell Signaling, Beverly, MA), and anti-Ki67 (Sigma-Aldrich, St. Louis, Missouri). The secondary antibodies were used. The immunohistochemical staining was evaluated in accordance with our previous report [34 ]. In brief, the proportion of tumor cells was graded as follows: 0 (no positive tumor cells), 1 (< 10% positive tumor cells), 2 (10-50% positive tumor cells), and 3 (> 50% positive tumor cells). The intensity of staining was determined as: 0 (no staining); 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). The staining index (SI) was calculated as staining intensity score × proportion of positive tumor cells, resulting in scores of 0, 1, 2, 3, 4, 6 and 9. An SI score of ≥ 4 was used to define tumors with high expression, and ≤ 3 as tumors with low expression.

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.

anti-p21^Cip1, anti-p27^Kip1, anti-CDK1, anti-CDK2, anti-CDK4, anti-cyclinA2, anti-cyclinB1, anti-cyclinD1, and anti-β tubulin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-SLC5A8 from Invitrogen (Carlsbad, CA, USA); goat anti-rabbit and goat anti-mouse antibodies were obtained from Bio-Rad (Hercules, CA, USA). CDK1/Cyclin B1, CDK2/Cyclin A2, CDK4/Cyclin D1, Retinoblastoma (C-term) and kinase buffer were purchased from SignalChem (Richmond, BC, Canada).