Journal: Molecular Medicine Reports

Article Title: Prognostic and predictive roles of microRNA-411 and its target STK17A in evaluating radiotherapy efficacy and their effects on cell migration and invasion via the p53 signaling pathway in cervical cancer

doi: 10.3892/mmr.2019.10826

Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction primer sequences.

Article Snippet: The membranes were incubated with phosphorylated (p)-STK17A antibody (cat. no. 14433-1-AP; Proteintech, Wuhan, China; 1:1,000), p-p21 WAF1 antibody (cat. no. AP01654PU-N; Origene; 1:10,000), p-p53 antibody (cat. no. MABE518; Merck KGaA; 1:1,000), TAp63 antibody (cat. no. TA311397; Origene; 1:1,000), and GAPDH antibody (cat. no. 10494-1-AP; Proteintech; 1:2,000) at 4°C overnight.

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Molecular Medicine Reports

Article Title: Prognostic and predictive roles of microRNA-411 and its target STK17A in evaluating radiotherapy efficacy and their effects on cell migration and invasion via the p53 signaling pathway in cervical cancer

doi: 10.3892/mmr.2019.10826

Figure Lengend Snippet: miR-411 activates the p53 signaling pathway and negatively regulates STK17A in cervical cancer cells. (A) Determination by reverse transcription-quantitative polymerase chain reaction analysis demonstrated that ectopic expression of miR-411 and siRNA-mediated knockdown of STK17A decreased the mRNA expression of STK17A, but increased the mRNA expression of p53, p21 WAF1 and TAp63; miR-411 inhibitor increased the mRNA expression of STK17A, but decreased the mRNA expression of p53, p21 WAF1 and TAp63. (B) Determination by western blot analysis and (C) quantification demonstrated that ectopic expression of miR-411 and siRNA-mediated knockdown of STK17A decreased the protein expression of STK17A, but increased the protein expression of p53, p21 WAF1 and TAp63; miR-411 inhibitor increased the protein expression of STK17A, but decreased the protein expression of p53, p21 WAF1 and TAp63. *P<0.05, vs. NC group; # P<0.05, vs. miR-411 inhibitor + siRNA-STK17A group. The experiment was repeated three times and data were compared by one-way analysis of variance and analyzed by Tukey's post hoc test. NC, negative control; miR-411, microRNA-411; STK17A, serine/threonine kinase 17a; siRNA, small interfering RNA.

Article Snippet: The membranes were incubated with phosphorylated (p)-STK17A antibody (cat. no. 14433-1-AP; Proteintech, Wuhan, China; 1:1,000), p-p21 WAF1 antibody (cat. no. AP01654PU-N; Origene; 1:10,000), p-p53 antibody (cat. no. MABE518; Merck KGaA; 1:1,000), TAp63 antibody (cat. no. TA311397; Origene; 1:1,000), and GAPDH antibody (cat. no. 10494-1-AP; Proteintech; 1:2,000) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Negative Control, Small Interfering RNA

Journal: Cell Death & Disease

Article Title: The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies

doi: 10.1038/cddis.2013.208

Figure Lengend Snippet: Brachyury modulates expression of cell cycle regulatory proteins. Western blot analysis of Brachyury and the cell cycle regulatory proteins Rb, cyclin D1 and p21 was performed using ( a ) the A549 tumor cell pair at indicated times after release from cell cycle arrest by serum starvation or ( b ) asynchronous cultures of H460 control versus Brachyury shRNA-1 and -2 cells. ( c ) Two tumor cell lines derived from single-cell cloning of H460 cells were analyzed for expression of Brachyury in relation to Rb and p21. ( d ) H460 cells transfected with p21 expression vector or pCMV control were analyzed for p21 and Rb expression by western blot and ( e ) growth kinetics over a 5-day period. ( f ) Indicated cells were treated with cytotoxic therapies and assayed for survival in comparison with untreated cells. ( g ) The H460 cell pair transfected with a pool of nonspecific control siRNA or p21-specific siRNAs was treated with γ -radiation (1 Gy) and evaluated for cell death by using CellTiter-Glo (Promega). (** P <0.01; *** P <0.001)

Article Snippet: The full-length human Brachyury construct (pCMV-Neo-Brachyury, accession number NM_003181.2), a p21 expression vector (accession number NM_078467.1) and empty-vector control (pCMV-Neo) were purchased from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Expressing, Western Blot, shRNA, Derivative Assay, Clone Assay, Transfection, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: The embryonic transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to conventional antitumor therapies

doi: 10.1038/cddis.2013.208

Figure Lengend Snippet: Brachyury drives repression of p21. ( a ) A ChIP assay was employed utilizing control IgG versus anti-Brachyury antibody with template DNA obtained from indicated cells. Shown at the top is the T-box palindromic consensus element juxtaposed with the half-binding site (shaded) located in the human p21 promoter (wild type), and a mutated p21 promoter containing point mutations in the T-box-binding site (denoted by arrowheads). Depicted below is the gel electrophoresis of PCR products amplified from the indicated immunoprecipitated DNA template sources using p21 promoter-specific primers. M corresponds to the DNA ladder. ( b ) Luciferase reporter assay was used to analyze H460 cells utilizing a wild-type p21 promoter driving the expression of the luciferase gene. ( c ) Luciferase reporter assay with H460 control.shRNA cells utilizing a wild-type versus mutated human p21 promoter driving the expression of the luciferase gene. Values shown represent the average of two technical replicates corrected for background±S.E.M. (* P <0.05). ( d ) Synchronized A549 pCMV and pBrachyury cells or asynchronous H460 control.shRNA and H460 Brachyury.shRNA2 were treated with indicated doses of radiation; cells collected after 24 h were analyzed for p21 induction by western blot. Intensity of p21 detection was quantitated and compared against β -actin or GAPDH (bottom panels)

Article Snippet: The full-length human Brachyury construct (pCMV-Neo-Brachyury, accession number NM_003181.2), a p21 expression vector (accession number NM_078467.1) and empty-vector control (pCMV-Neo) were purchased from Origene Technologies Inc. (Rockville, MD, USA).

Techniques: Binding Assay, Nucleic Acid Electrophoresis, Amplification, Immunoprecipitation, Luciferase, Reporter Assay, Expressing, shRNA, Western Blot

Journal: PLoS ONE

Article Title: The Role of Dlc1 Isoform 2 in K-Ras2 G12D Induced Thymic Cancer

doi: 10.1371/journal.pone.0040302

Figure Lengend Snippet: A(i) p21 waf1 protein in T-cell lymphoma and carcinoma cell lines, (ii) Plot showing the relative intensity of p21 waf1 protein in T-cell lymphoma and carcinoma cell lines relative to actin. Means and standard error of mean were determined from at least five independent experiments. (*p<0.05, **P<0.01, ***P<0.001, ****P<.0001). (B) Measurement of active RhoGTP in cell lines . (i) RhoGTP pull down assay showing constitutive and LPA induced levels of active RhoGTP in T-cell lymphoma and thymic epithelial carcinoma cell lines. (ii) Plot showing the RhoGTP to total Rho protein ratio by scanning the relative intensity of the protein bands in non-treated and LPA induced in TEC, TL, MEF cell lines. Means and standard error of mean determined from four independent experiments. (*p<0.05, **P<0.01, ***P<0.001 and ****P<.0001 by Student t test and two way ANOVA test.

Article Snippet: The western blots were hybridized with Dlc1 antibody (Sc32931, Santa Cruz Biotechnology, CA, USA) and p21 waf1 antibody (Acris Antibodies Gbmh, AP06263PU-N) and visualized.

Techniques: Pull Down Assay

Journal: Scientific Reports

Article Title: Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery

doi: 10.1038/srep38421

Figure Lengend Snippet: ( A , B ) THP-1 cells were transfected with TM siRNA or HA-TM FL plasmid for 24 h, which was followed by PMA stimulation for 72 h. The expression of p21 Cip1/WAF1 and PCNA in total cell lysates was assayed using western blot analysis. ( C , upper and D , upper) THP-1 cells were stimulated with 150 nM PMA for 24–72 h.The expression of phosphorylated ERK1/2 in total cell lysates and NF-κB p65 in nuclear fraction were assayed using western blot analysis. ( C , lower and D , lower), THP-1 cells were transfected with TM siRNA or HA-TM FL plasmid for 24 h, which was followed by PMA stimulation for 72 h. The expression of phosphorylated ERK1/2 and NF-κB p65 was assayed using western blot analysis. ( E ) THP-1 cells were pretreated with 10 μM PD98059 for 1 h followed by PMA treatment (dark gray) or treated with PD98059 only (white) for 72 h (black: naïve THP-1 cells; light gray: PMA treatment). A western blot assay was performed to determine the expression of NF-kB p65 (nuclear fraction), PCNA, and p21 Cip1/WAF1 (total cell lysates). Lamin B1, total ERK1/2, and β-actin were used as the loading controls, as indicated. Five independent experiments have been performed (n = 5) for all studies, and representative images have been showed. The density of each band was quantified using densitometry and related protein expression was presented as bar graph. The data are presented as the mean ± SD, and * p < 0.05 was considered significant.

Article Snippet: The membranes were respectively probed with rabbit anti-human TM (TA307719, OriGene Technologies, Inc, Rockville, MD, USA), rabbit anti- p21 Cip1/WAF1 (#2947, Cell Signaling, Danvers, CA, USA), rabbit anti-PCNA (sc-7907, Santa Cruz biotechnology, Santa Cruz, CA, USA), mouse anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9106, Cell Signaling, Danvers, CA, USA), rabbit anti-p44/42 MAPK (Erk1/2)(#9102, Cell Signaling, Danvers, CA, USA), rabbit anti-phospho-NF-κB p65 (Ser536)(#3033, Cell Signaling, Danvers, CA, USA), rabbit anti-Paxillin (phospho Y31)(ab32115, Abcam, MA, USA), rabbit anti-Paxillin (ab32084), rabbit anti-Cofilin (phospho S3)(ab47281, Abcam, MA, USA), rabbit anti-Cofilin (ab54532, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (phospho T508)(ab38508, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (ab87971, Abcam, MA, USA), rabbit anti-PYK2 (phospho Y881)(ab4801, Abcam, MA, USA), rabbit anti-PYK2 (ab32571, Abcam, MA, USA), mouse anti-PKC α (sc-8393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PKC βII (sc-210, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-PKC θ (sc-1875, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti- PKCδ (sc-937-G, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-PKC ε (sc-214, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery

doi: 10.1038/srep38421

Figure Lengend Snippet: ( A ) THP-1 cells were transfected with 2 or 4 μg of PKCα, PKCβ, PKCδ, PKCε, or PKCθ shRNA for 24 h. Total cell lysates were purified, and knockdown efficiency was assayed using western blot analysis. ( B ) The THP-1 cells were knocked down by PKCα, PKCβ, PKCδ, PKCε, and PKCθ shRNAs for 24 h followed by PMA stimulation for 72 hours. The number of CD14 + cells was scored using flow cytometry. Data are expressed as a % of the control, are presented as the mean ± SD and represent the results of five independent experiments (n = 5, * p < 0.05 was considered significant). ( C ) Different sets of THP-1 cells were transfected with 4 μg of each shRNA for 24 h followed by PMA stimulation for 72 hours. The levels of p21 Cip1/WAF1 and PCNA were analyzed using western blot analysis. ( D ) The THP-1 cells were knocked down by PKCδ shRNA for 24 h followed by PMA stimulation for 72 hours. The level of PKCδ and total and phosphorylated ERK1/2 was analyzed using western blot analysis. In western blot analysis, β-actin and total-ERK1/2 were used as loading controls. The density of each band was quantified using densitometry and related protein expression was presented as bar graph. The data are presented as the mean ± SD, and * p < 0.05 was considered significant (n = 5). ( E ) Lysates of THP-1 cells with PMA stimulation were extracted. Left, immunoprecipitated using goat anti-PKCδ or goat IgG control antibodies followed by western blot analysis and detected using goat anti-PKCδ or rabbit anti-TM antibodies. Right, immunoprecipitated using rabbit anti-TM or rabbit IgG control antibodies followed by western blot analysis and detected using rabbit anti-TM or goat anti-PKCδ antibodies. Five independent experiments have been performed (n = 5). ( F ) THP-1 cells were treated without (upper photos) or with (lower photos) PMA for 72 hours. Subcellular distributions of TM (triangle) and PKCδ (arrow) in THP-1 cells were detected by immunofluorescence and observed by confocal microscopy. DAPI was used to stain the nuclei of THP-1 cells.

Article Snippet: The membranes were respectively probed with rabbit anti-human TM (TA307719, OriGene Technologies, Inc, Rockville, MD, USA), rabbit anti- p21 Cip1/WAF1 (#2947, Cell Signaling, Danvers, CA, USA), rabbit anti-PCNA (sc-7907, Santa Cruz biotechnology, Santa Cruz, CA, USA), mouse anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9106, Cell Signaling, Danvers, CA, USA), rabbit anti-p44/42 MAPK (Erk1/2)(#9102, Cell Signaling, Danvers, CA, USA), rabbit anti-phospho-NF-κB p65 (Ser536)(#3033, Cell Signaling, Danvers, CA, USA), rabbit anti-Paxillin (phospho Y31)(ab32115, Abcam, MA, USA), rabbit anti-Paxillin (ab32084), rabbit anti-Cofilin (phospho S3)(ab47281, Abcam, MA, USA), rabbit anti-Cofilin (ab54532, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (phospho T508)(ab38508, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (ab87971, Abcam, MA, USA), rabbit anti-PYK2 (phospho Y881)(ab4801, Abcam, MA, USA), rabbit anti-PYK2 (ab32571, Abcam, MA, USA), mouse anti-PKC α (sc-8393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PKC βII (sc-210, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-PKC θ (sc-1875, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti- PKCδ (sc-937-G, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-PKC ε (sc-214, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Transfection, shRNA, Purification, Western Blot, Flow Cytometry, Expressing, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Staining

Journal: Scientific Reports

Article Title: Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery

doi: 10.1038/srep38421

Figure Lengend Snippet: ( A ) Human PBMCs were stimulated with 150 nM PMA for 12–48 h. Upper, the expression of phosphorylated ERK1/2 in total cell lysates were assayed using western blot analysis. Lower, human PBMCs were treated with 150 nM PMA, pretreated with 10 μM PD98059 for 1 h followed by PMA treatment, or treated with PD98059 alone for 48 h. A western blot assay was performed to determine the expression of p21 Cip1/WAF1 . The total-ERK1/2 and β-actin were used as loading controls. Three independent experiments (PBMCs from 3 voluntary donors) have been performed (n = 3), and representative images have been showed. The density of each band was quantified using densitometry and related protein expression was presented as bar graph. The data are presented as the mean ± SD, and * p < 0.05 was considered significant. ( B ) Lysates of PBMCs with 150 nM PMA stimulation for 24 hours were extracted. Upper, immunoprecipitated using goat anti-PKCδ or goat IgG control antibodies followed by western blot analysis and detected using goat anti-PKCδ or rabbit anti-TM antibodies. Lower, immunoprecipitated using rabbit anti-TM or rabbit IgG control antibodies followed by western blot analysis and detected using rabbit anti-TM or goat anti-PKCδ antibodies. ( C ) PBMCs were treated with 150 nM PMA for 24 hours. The expression of CD68 in PBMCs was detected by immunofluorescence and observed by confocal microscopy. ( D ) PBMCs were treated without (upper photos) or with (lower photos) PMA for 24 hours. Subcellular distributions of TM (arrow) and PKCδ (arrow) in PBMCs were detected by immunofluorescence and observed by confocal microscopy. DAPI was used to stain the nuclei of PBMCs. Three independent experiments (PBMCs from 3 voluntary donors) have been performed, and representative images were presented.

Article Snippet: The membranes were respectively probed with rabbit anti-human TM (TA307719, OriGene Technologies, Inc, Rockville, MD, USA), rabbit anti- p21 Cip1/WAF1 (#2947, Cell Signaling, Danvers, CA, USA), rabbit anti-PCNA (sc-7907, Santa Cruz biotechnology, Santa Cruz, CA, USA), mouse anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9106, Cell Signaling, Danvers, CA, USA), rabbit anti-p44/42 MAPK (Erk1/2)(#9102, Cell Signaling, Danvers, CA, USA), rabbit anti-phospho-NF-κB p65 (Ser536)(#3033, Cell Signaling, Danvers, CA, USA), rabbit anti-Paxillin (phospho Y31)(ab32115, Abcam, MA, USA), rabbit anti-Paxillin (ab32084), rabbit anti-Cofilin (phospho S3)(ab47281, Abcam, MA, USA), rabbit anti-Cofilin (ab54532, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (phospho T508)(ab38508, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (ab87971, Abcam, MA, USA), rabbit anti-PYK2 (phospho Y881)(ab4801, Abcam, MA, USA), rabbit anti-PYK2 (ab32571, Abcam, MA, USA), mouse anti-PKC α (sc-8393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PKC βII (sc-210, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-PKC θ (sc-1875, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti- PKCδ (sc-937-G, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-PKC ε (sc-214, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Expressing, Western Blot, Immunoprecipitation, Immunofluorescence, Confocal Microscopy, Staining

Journal: Scientific Reports

Article Title: Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery

doi: 10.1038/srep38421

Figure Lengend Snippet: TM expression is increased soon after PMA induction in THP-1 cells. TM may regulate PMA-induced THP-1 cell differentiation via a direct interaction with PKCδ. TM acts as a scaffold for PKCδ docking, which keeps PKCδ in the region close to the membrane and promotes subsequent ERK1/2 activation. ERK1/2 activation subsequently enhances the expression of cell cycle inhibitor p21 Cip1/WAF1 via NF-kB p65 signaling, which causes cell cycle arrest and promotes differentiation. On the other hand, ERK1/2 activation participates in the phosphorylation of paxillin, cofilin, LIMK1, and PYK2, which interact with TM and mediate cytoskeleton remodeling to promote differentiation.

Article Snippet: The membranes were respectively probed with rabbit anti-human TM (TA307719, OriGene Technologies, Inc, Rockville, MD, USA), rabbit anti- p21 Cip1/WAF1 (#2947, Cell Signaling, Danvers, CA, USA), rabbit anti-PCNA (sc-7907, Santa Cruz biotechnology, Santa Cruz, CA, USA), mouse anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (#9106, Cell Signaling, Danvers, CA, USA), rabbit anti-p44/42 MAPK (Erk1/2)(#9102, Cell Signaling, Danvers, CA, USA), rabbit anti-phospho-NF-κB p65 (Ser536)(#3033, Cell Signaling, Danvers, CA, USA), rabbit anti-Paxillin (phospho Y31)(ab32115, Abcam, MA, USA), rabbit anti-Paxillin (ab32084), rabbit anti-Cofilin (phospho S3)(ab47281, Abcam, MA, USA), rabbit anti-Cofilin (ab54532, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (phospho T508)(ab38508, Abcam, MA, USA), rabbit anti-LIM Kinase 1 (ab87971, Abcam, MA, USA), rabbit anti-PYK2 (phospho Y881)(ab4801, Abcam, MA, USA), rabbit anti-PYK2 (ab32571, Abcam, MA, USA), mouse anti-PKC α (sc-8393, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-PKC βII (sc-210, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti-PKC θ (sc-1875, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat anti- PKCδ (sc-937-G, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-PKC ε (sc-214, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies.

Techniques: Expressing, Cell Differentiation, Activation Assay

Journal: Oncotarget

Article Title: Decreased DACH1 expression in glomerulopathy is associated with disease progression and severity

doi: 10.18632/oncotarget.13470

Figure Lengend Snippet: A representative micrograph of double immunofluorescence was performed for DACH1(red) ( B , E , H , K ), P21 (green) ( A ), P53(green) ( D ), Cyclin A (green) ( G ), Cyclin D1 (green) ( J ), Merge images (yellow) ( C , F , I , L ). Original magnification is ×400, Bar, 50 μm.

Article Snippet: DACH1 rabbit polyclonal antibody (1:1000 dilution, Proteintech, Wuhan, China), Cyclin D1, PCNA mouse monoclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, Dallas, TX), Cyclin A, Cyclin B1, p21, p53 rabbit polyclonal antibody (1:500, Affinity Biosciences, USA) were used as primary antibodies.

Techniques: Immunofluorescence

Journal: Oncotarget

Article Title: Decreased DACH1 expression in glomerulopathy is associated with disease progression and severity

doi: 10.18632/oncotarget.13470

Figure Lengend Snippet: ( A ) Representative Western blot images and ( B ) summarized data showing DACH1, p21, p53, CyclinD1, CyclinA expression with Plasmid. DACH1-transfected HK2. ( C ) representative Western blot images and ( E ) summarized data showing that the abundance DACH1, p21, p53 were increased in Plasmid. DACH1-transfected podocytes cultured in a permissive temperature (33°C). ( D ) representative Western blot images and ( F ) summarized data showing that the abundance p21, p53 were increased, while CyclinD1 was decreased in Plasmid. DACH1-transfected podocytes cultured in a permissive temperature (37°C). * P < 0.05 vs. Null-HK2 or Null-podocyte. All experiment were performed at least thrice with samples from independent experiments.

Article Snippet: DACH1 rabbit polyclonal antibody (1:1000 dilution, Proteintech, Wuhan, China), Cyclin D1, PCNA mouse monoclonal antibody (1:1000 dilution, Santa Cruz Biotechnology, Dallas, TX), Cyclin A, Cyclin B1, p21, p53 rabbit polyclonal antibody (1:500, Affinity Biosciences, USA) were used as primary antibodies.

Techniques: Western Blot, Expressing, Plasmid Preparation, Transfection, Cell Culture