Anti p27

Immunoprecipitation assays were performed as previously described [31 (link)]. Cells were washed twice with PBS, collected and homogenized with RIPA buffer. After cell debris was removed by centrifugation, extracts were aliquoted and either used immediately or stored at -80°C. Whole-cell lysates in lysis buffer were cleared with 1.0 μg nonimmune rabbit IgG (Santa Cruz) together with 30 μl of protein A-Sepharose beads (Pierce). After centrifugation, the lysates were immunoprecipitated for 1 h at 4°C with 1 μg of the anti-Stat6 antibody or nonimmune rabbit IgG and then incubated overnight at 4°C with protein A-Sepharose. The immunoprecipitates were washed three times with lysis buffer and once with PBS and then resuspended in electrophoresis sample buffer. Samples of immunoprecipitated or total proteins (30 μg) were analyzed by Western blotting using the anti-ppRB-Ser807/811 antibody (Cell Signaling Technology) against a pRB peptide phosphorylated on the Ser807/811 residue, which is phosphorylated by both CDK2 and CDK4/6 kinases [32 ], or the anti-pRB against underphosphorylated pRB (BD Biosciences-Pharmingen), the anti-PR antibody (abcam), anti-p21(abcam), anti-p27(abcam), and anti-GADPH (as control antibody). The blots represent typical results from at least three independent experiments.

Total cell extracts were prepared using Pierce IP lysis buffer (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amounts of proteins were subjected to WB analysis. The antibodies used for WBs were anti-β-Actin (mouse, Sigma, USA), anti-CUL1 (rabbit, Thermo Fisher Scientific, USA), anti-CUL2 (mouse, Santa Cruz Biotechnology, USA), anti-CUL3 (rabbit, Cell Signaling Technology, USA), anti-CUL4A (mouse, Sigma, USA), anti-CUL4B (mouse, Sigma, USA), anti-CUL5 (rabbit, Abcam, USA), anti-CUL7 (rabbit, OriGene, USA), anti-Flag (mouse, Sigma, USA), anti-RBX1 (rabbit, Cell Signaling Technology, USA), anti-DCAF1 (rabbit, Abcam, USA), anti-DCAF4 (rabbit, Sigma, USA), anti-DCAF8 (rabbit, Sigma, USA), anti-DCAF11 (rabbit, Sigma, USA), anti-DCAF15 (rabbit, Sigma, USA), anti-DDB1 (rabbit, Sigma, USA), anti-p21 (rabbit, Sigma, USA), and anti-p27 (mouse, Abcam, USA). Signals from WBs were recorded using a ChemiDoc MP (Bio-Rad, USA). All experiments were performed in triplicate.

Protein lysates of ESCC cells were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22-mm NC membranes (Sigma), and incubated with specific antibodies: anti-CDK6, anti-CDK4, anti-CDK2, anti-cyclinD1, anti-cyclinD3, anti-p27, anti-p21, anti-CDKN2C (Abcam, Shanghai, China), anti-EZH2, anti-EED, anti-SUZ12, anti-EZH1, and anti-β-actin (Cell Signaling Technology). The dilution ratio of the primary antibodies was 1:1,000, although anti-β-actin was diluted to 1:8,000 for Western blotting. Protein bands were visualized with Super Signal Chemiluminescence Substrate (Thermo Scientific) and β-actin was used as a control.

Western blot analysis was performed as described previously^{40 (link)}. The following primary antibodies were used: anti-cyclin D1 (60186-lg, Proteintech, Chicago, IL, USA), anti-p27 (ab193379, Abcam, Cambridge, UK), anti-p21 (#2947, Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated Rb (Ser795) (#9301, Cell Signaling Technology), anti-IRS1 (#2382, Cell Signaling Technology), anti-phosphorylated IRS1 (Ser307) (D151214, BBI, Shanghai, China), anti-IGF1R (20254-1-AP, Proteintech), anti-phosphorylated AKT (Ser473) (66444-1-lg, Proteintech), anti-AKT (10176-2-AP, Proteintech), anti-phosphorylated mTOR (Ser2448) (D155324, BBI), anti-mTOR (20657-1-AP, Proteintech), anti-FoxP3 (D260367, BBI), anti-Dicer (ab14601, Abcam), anti-Drosha (55001-1-AP, Proteintech), and anti-GAPDH (#2118, Cell Signaling Technology).

After treatment with HDACIs, the cells were lysed with lysis buffer (Cell Signaling, Danvers, MA) including a protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany). Western blot analysis was performed as previously reported [40 (link)]. Antibodies used for immunodetection were anti-p21 (1:1,000, Abcam, Cambridge, MA), anti-p27 (1:500, Abcam), anti-CDK2 (1:500, Thermo Scientific, Rockford, IL), anti-CDK4 (1:200, Abcam), anti-acetyl H3 (K9 + K14 + K18 + K23 + K27, 1:1,000, Abcam), anti-acetyl H3 (K27,1:2,000, Abcam), anti-acetyl H4 (K5 + K8 + K12 + K16,1:2,000, Abcam) and anti-β-actin (1:10,000, Sigma-Aldrich).
After the blot was washed, it was incubated with a horseradish peroxidase-conjugated species-specific secondary antibody (1:5,000, Jackson Laboratory, West Grove, PA) for 1 h at room temperature. The blots were developed using a chemiluminescence detection system (Invitrogen, Carlsbad, CA). Band density was analyzed using NIH ImageJ software. Densitometric measurements were performed on individual immunoblot for each antibody tested, and the values indicate the protein level normalized to the corresponding β-actin levels.

The analysis of protein expression were performed as previously published [15 (link), 16 (link)]. In brief, the Hepa1c1c7 cells (1 × 10⁵ cells/well, the cell density reached 80 to 90% confluence), were treated with PHO-S (0.3–2.0 mM), DODAC/PHO-S 1:1 (0.3–2.0 mM), and empty DODAC (0.3–2.0 mM), for 12 h, were washed with PBS and resuspended in FACS buffer with 2.5% paraformaldehyde for 1 h. After washing, cells were again resuspended in a primary antibody specific for the proteins anti-CD44, anti-CD90, anti-p53, anti-p21, anti-p27, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-caspase-8 (Abcam, Cambridge, MA, United States); anti- cytochrome c, anti-DR4 (Santa (Cruz Biotechnology Inc., Santa Cruz, EUA) and anti-cyclin D1 (Cell Signaling Technology, Danvers, MA), at a concentration of 1 μg/ml at 4 °C, for 1 min. The corresponding isotope antibody was used as a negative control and as a secondary antibody was used Goat anti Mouse IgG (H/L): FITC (AbD Serotec, Raleigh, NC, United States). The cells were pelleted, washed twice with PBS, then, fluorescence-activated cell sorting (FACS) analysis was performed on BD Biosciences FACs Calibur flow cytometer (Becton Dickinson, San Jose, CA, United States) using Cell Quest and Win MDI 2.9 softwares.

For western blot analysis, proteins were separated in a 12% SDS-polyacrylamide gel. After electrophoresis, the gel was transferred onto PVDF membranes (Millipore, USA) and blocked for 3 h at 37 °C with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Primary antibodies were incubated at 4 °C overnight. Primary antibodies were used at the following dilutions: anti-p27 (Abcam, USA) at 1:2000, anti-p21 (Abcam, USA) at 1:2000, anti-p53 (Abcam, USA) and anti-phosphorylation of p53 at 1:2000, and anti-GAPDH (Abcam, USA) at 1:10,000. After primary antibody probing, membranes were washed in TBST, and incubated with HRP-conjugated secondary antibody (Dako, Denmark) at 1:5000 for 60 min at room temperature. After further washing, protein expression was detected by enhanced chemiluminescent (ECL) substrate (Pierce, Thermo Fisher Scientific, USA) and protein bands were visualized by film exposure. GAPDH was used as an internal control. The immunoreactive density was analyzed by Quantity One (Bio-Rad, USA).