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Cell Signaling Technology Inc p21
P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Autoradiography:

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling). .. The blots were visualized using Pierce ECL Western Blotting Substrates (32106/34080/34096, Thermo, Rockford, IL) and HyBlot CL Autoradiography Film (E3018, Denville Scientific, Metuchen, NJ).

Cytometry:

Article Title: L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q-syndrome in a TP53-independent way
Article Snippet: Paragraph title: Flow cytometry ... Cells were incubated with primary antibody for CDKN1A (#12D1; Cell Signaling Technology, Beverly, MA) followed by anti-rabbit antibody AlexaFluor 750 (Molecular Probes, Grand Island, NY).

Blocking Assay:

Article Title: Combined autophagy and HDAC inhibition
Article Snippet: .. Slides were exposed to MAP1LC3B (Abcam, AB48394), CDKN1A (Cell Signaling Technologies, 2947), SQSTM1 (Abcam, AB91526), MKI67 (Cell Signaling Technologies, 9027), active CASP3 (Cell Signaling Technologies, 9661) and CTSD (Abcam, AB91526) antibodies diluted in the protein block solution at 4 °C overnight as previously described. .. After washing with PBS, slides were incubated in the appropriate secondary antibody (Jackson Immunoresearch, 111-035-003) for 1 h at ambient temperature.

Article Title: SOX2 suppresses CDKN1A to sustain growth of lung squamous cell carcinoma
Article Snippet: After being autoclaved in target retrieval solution (Dako, Carpinteria, CA, USA) for 15 minutes, sections were incubated with 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase activity. .. A primary antibody specific for human SOX2 and CDKN1A was obtained from Cell Signaling Technology (Beverly, MA).

Electrophoresis:

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: Total cell lysates containing equal amounts of protein were subjected to 4–20% mini-TGX gel (456–1096, Bio-Rad, Hercules, CA) electrophoresis and then transferred to PVDF membranes. .. Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling).

Incubation:

Article Title: Baicalein inhibits pancreatic cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream Akt and ERK signaling pathways
Article Snippet: The membranes were incubated with the primary antibodies and then the corresponding secondary antibodies (horseradish peroxidase-conjugated goat anti-Rabbit IgG or goat anti-mouse IgG at 1:1000 dilution, Beyotime Biotechnology). .. Primary antibodies used: NEDD9 (#4044, 1:500), Akt (#4691, 1:1000), p-Akt S-473 (#4060, 1:1000), p-Akt T-308 (#2965, 1:1000), ERK1/2 (#9102, 1:1000), p-ERK1/2 (#9101, 1:1000), PDK1(#3062, 1:1000), P21(#8831, 1:1000), P27(#3686, 1:1000) and cleaved caspase-9 (#7237, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA), Bax (AB026, 1:1000) and Bcl-2 (AB112, 1:1000) from Beyotime Biotechnology (Shanghai, China).

Article Title: L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q-syndrome in a TP53-independent way
Article Snippet: .. Cells were incubated with primary antibody for CDKN1A (#12D1; Cell Signaling Technology, Beverly, MA) followed by anti-rabbit antibody AlexaFluor 750 (Molecular Probes, Grand Island, NY). .. Flow cytometry was performed on a FACSCanto cytometer (BD Biosciences, San Jose, CA) and data were analysed using FlowJo software version 8.7.1 (TreeStar, Ashland, OR).

Article Title: A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region
Article Snippet: The blots were blocked with 3% BSA for 2 h at 25°C and then probed with primary antibodies (1:1,000) at 4°C for 16 h. After three washes, the blots were subsequently incubated with the corresponding secondary antibodies (1:3,000) for 2 h at 25°C. .. Antibodies to β-actin (#4970, Cell Signaling Technology, MA, USA), Phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), Phospho-p53 (Ser15) (#9286, Cell Signaling Technology), γH2AX (#9718, Cell Signaling Technology), C-MYC (#9402, Cell Signaling Technology), P21(#2947, Cell Signaling Technology), P27(#3686, Cell Signaling Technology), anti-rabbit IgG-HRP (#7074, Cell Signaling Technology), and anti-mouse IgG-HRP (#7076, Cell Signaling Technology) were used.

Article Title: Combined autophagy and HDAC inhibition
Article Snippet: Following this, slides were incubated in a protein block solution (5% horse serum and 1% goat serum (Gibco, 16050 and 16210) in PBS (Corning Cellgro, 21-031-CV) for 20 min. .. Slides were exposed to MAP1LC3B (Abcam, AB48394), CDKN1A (Cell Signaling Technologies, 2947), SQSTM1 (Abcam, AB91526), MKI67 (Cell Signaling Technologies, 9027), active CASP3 (Cell Signaling Technologies, 9661) and CTSD (Abcam, AB91526) antibodies diluted in the protein block solution at 4 °C overnight as previously described.

Article Title: Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells
Article Snippet: .. After transfer in cold room, the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies: beta-actin(1:3000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1(1:1000), CDK4(1:1000), CDK6(1:1000), p27 (1:1000) and p21(1:1000) (Cell Signaling, Boston, MA, USA). .. After incubation with peroxidase-coupled anti-mouse IgG and anti-rabbit IgG (Santa Cruz Biotechnology) at 37°C for 1 hour, bound proteins were visualized using ECL (Thermo Fisher Scientific) and detected using x films.

Article Title: SOX2 suppresses CDKN1A to sustain growth of lung squamous cell carcinoma
Article Snippet: After being autoclaved in target retrieval solution (Dako, Carpinteria, CA, USA) for 15 minutes, sections were incubated with 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase activity. .. A primary antibody specific for human SOX2 and CDKN1A was obtained from Cell Signaling Technology (Beverly, MA).

Article Title: Network Pharmacology Integrated Molecular Docking Reveals the Antiosteosarcoma Mechanism of Biochanin A
Article Snippet: .. Each group of membrane was subsequently treated with dilutions of primary antibody specific for BGLAP (Abcam, Cambridge, USA), ATF3 (Abcam, Cambridge, USA), BAX (Cell Signaling Technology, Danvers, USA), CDKN1A (Cell Signaling Technology, Danvers, USA), and TP53 (Cell Signaling Technology, Danvers, USA) or endogenous reference GAPDH and incubated at 4°C overnight. .. After that, it was washed for 3 times with TBST and treated with horseradish peroxidase conjugated secondary antibodies for 1-hour incubation at room temperature.

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: .. Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling). .. The blots were visualized using Pierce ECL Western Blotting Substrates (32106/34080/34096, Thermo, Rockford, IL) and HyBlot CL Autoradiography Film (E3018, Denville Scientific, Metuchen, NJ).

Activity Assay:

Article Title: SOX2 suppresses CDKN1A to sustain growth of lung squamous cell carcinoma
Article Snippet: After being autoclaved in target retrieval solution (Dako, Carpinteria, CA, USA) for 15 minutes, sections were incubated with 3% hydrogen peroxide for 5 minutes to block endogenous peroxidase activity. .. A primary antibody specific for human SOX2 and CDKN1A was obtained from Cell Signaling Technology (Beverly, MA).

Expressing:

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: Immunoblot analysis Immunoblots were performed to determine the relative expression of AR, cyclins, CDKs, CDKN1A, and phosphorylation of RB protein at various time points after treatment with 10 nM DHT or vehicle control. .. Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling).

BIA-KA:

Article Title: Baicalein inhibits pancreatic cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream Akt and ERK signaling pathways
Article Snippet: The concentrations of the total proteins were measured using the Bicinchoninic acid (BCA) method (Beyotime Biotechnology). .. Primary antibodies used: NEDD9 (#4044, 1:500), Akt (#4691, 1:1000), p-Akt S-473 (#4060, 1:1000), p-Akt T-308 (#2965, 1:1000), ERK1/2 (#9102, 1:1000), p-ERK1/2 (#9101, 1:1000), PDK1(#3062, 1:1000), P21(#8831, 1:1000), P27(#3686, 1:1000) and cleaved caspase-9 (#7237, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA), Bax (AB026, 1:1000) and Bcl-2 (AB112, 1:1000) from Beyotime Biotechnology (Shanghai, China).

Article Title: Up-Regulation of MicroRNA-21 Correlates with Lower Kidney Cancer Survival
Article Snippet: Protein assays were performed using a BCA Protein assay kit (Pierce/Thermo Scientific, Rockford, IL, USA) according to the manufacturer's instructions. .. All antibodies, which included p21(cat. no. 2552), p38 MAPkinase (cat. no. 9212) and cyclinE2 (cat. no. 4132) were purchased from Cell Signaling (Danvers, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Article Title: A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region
Article Snippet: Western blotting The cells treated with compounds or control medium were collected and lysed in RIPA lysis buffer (Bioteke, China), and the protein concentrations were determined using a BCA protein assay kit (Pierce, U.S.A.). .. Antibodies to β-actin (#4970, Cell Signaling Technology, MA, USA), Phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), Phospho-p53 (Ser15) (#9286, Cell Signaling Technology), γH2AX (#9718, Cell Signaling Technology), C-MYC (#9402, Cell Signaling Technology), P21(#2947, Cell Signaling Technology), P27(#3686, Cell Signaling Technology), anti-rabbit IgG-HRP (#7074, Cell Signaling Technology), and anti-mouse IgG-HRP (#7076, Cell Signaling Technology) were used.

Article Title: Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells
Article Snippet: Western blot analysis Total proteins from SKOV3 and Hey cells were extracted in lysis buffer (Thermo Fisher Scientific, Rockford, IL) and quantified using the BCA method. .. After transfer in cold room, the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies: beta-actin(1:3000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1(1:1000), CDK4(1:1000), CDK6(1:1000), p27 (1:1000) and p21(1:1000) (Cell Signaling, Boston, MA, USA).

Western Blot:

Article Title: Targeting Polo-like kinase 1 in SMARCB1 deleted atypical teratoid rhabdoid tumor
Article Snippet: .. Antibodies used for western blot analysis were from the following sources: actin #8H10D10, p21#2947, Rad 51#8875, 53BP1#4937, Plk1#5472, ki67# 9449, KU80#2753, Phospho-H2AX#2577 were purchased from Cell Signaling, USA; p53 ab179477 from abcam, INI-1(Anti-BAF47) from BD Biosciences, USA. .. BT12, BT16 atypical teratoid/rhabdoid tumor (ATRT) cell lines were kindly provided by Dr. Peter Houghton’s laboratory (St. Jude Children’s Research Hospital).

Article Title: Baicalein inhibits pancreatic cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream Akt and ERK signaling pathways
Article Snippet: Paragraph title: Western blotting ... Primary antibodies used: NEDD9 (#4044, 1:500), Akt (#4691, 1:1000), p-Akt S-473 (#4060, 1:1000), p-Akt T-308 (#2965, 1:1000), ERK1/2 (#9102, 1:1000), p-ERK1/2 (#9101, 1:1000), PDK1(#3062, 1:1000), P21(#8831, 1:1000), P27(#3686, 1:1000) and cleaved caspase-9 (#7237, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA), Bax (AB026, 1:1000) and Bcl-2 (AB112, 1:1000) from Beyotime Biotechnology (Shanghai, China).

Article Title: Up-Regulation of MicroRNA-21 Correlates with Lower Kidney Cancer Survival
Article Snippet: Paragraph title: Western analysis ... All antibodies, which included p21(cat. no. 2552), p38 MAPkinase (cat. no. 9212) and cyclinE2 (cat. no. 4132) were purchased from Cell Signaling (Danvers, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Article Title: Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/β-Catenin Signaling Pathway in Glioma Cells
Article Snippet: Paragraph title: 4.5. Western Blot Analysis ... The following primary antibodies were used: phospho-AMPK (Thr-172; 1:1000; 2535S), AMPK (1:1000; 2532S), c-caspase-3 (1:1000; 9664S), caspase-3 (1:1000; 9662S), Beclin1 (1:1000; 3495S), Bax (1:1000; 2772S), p21(1:1000; 2947S), Bcl-xL (1:1000; 2764S), and phospho-P53 (p15; 1:1000; 9284S), obtained from Cell Signaling.

Article Title: A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region
Article Snippet: Paragraph title: Western blotting ... Antibodies to β-actin (#4970, Cell Signaling Technology, MA, USA), Phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), Phospho-p53 (Ser15) (#9286, Cell Signaling Technology), γH2AX (#9718, Cell Signaling Technology), C-MYC (#9402, Cell Signaling Technology), P21(#2947, Cell Signaling Technology), P27(#3686, Cell Signaling Technology), anti-rabbit IgG-HRP (#7074, Cell Signaling Technology), and anti-mouse IgG-HRP (#7076, Cell Signaling Technology) were used.

Article Title: Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells
Article Snippet: Paragraph title: Western blot analysis ... After transfer in cold room, the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies: beta-actin(1:3000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1(1:1000), CDK4(1:1000), CDK6(1:1000), p27 (1:1000) and p21(1:1000) (Cell Signaling, Boston, MA, USA).

Article Title: Network Pharmacology Integrated Molecular Docking Reveals the Antiosteosarcoma Mechanism of Biochanin A
Article Snippet: Paragraph title: 2.9. Western-Blot Assay ... Each group of membrane was subsequently treated with dilutions of primary antibody specific for BGLAP (Abcam, Cambridge, USA), ATF3 (Abcam, Cambridge, USA), BAX (Cell Signaling Technology, Danvers, USA), CDKN1A (Cell Signaling Technology, Danvers, USA), and TP53 (Cell Signaling Technology, Danvers, USA) or endogenous reference GAPDH and incubated at 4°C overnight.

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: Immunoblot analysis Immunoblots were performed to determine the relative expression of AR, cyclins, CDKs, CDKN1A, and phosphorylation of RB protein at various time points after treatment with 10 nM DHT or vehicle control. .. Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling).

Article Title: Treatment of melanoma with selected inhibitors of signaling kinases effectively reduces proliferation and induces expression of cell cycle inhibitors
Article Snippet: Paragraph title: Western blot analysis ... They used to analyze the following antibodies: Cyclin D1 (#2926 Cell Signaling TM), cyclin D3 (#2936 Cell Signaling TM), CDK4 kinase (#2906 Cell Signaling TM), CDK6 kinase (#3136 Cell Signaling TM), p16 (#4824 Cell Signaling TM), p21(#2946 Cell Signaling TM), p27 (#2552 Cell Signaling TM), and β-actin (A2228, SIGMA).

Flow Cytometry:

Article Title: L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q-syndrome in a TP53-independent way
Article Snippet: Paragraph title: Flow cytometry ... Cells were incubated with primary antibody for CDKN1A (#12D1; Cell Signaling Technology, Beverly, MA) followed by anti-rabbit antibody AlexaFluor 750 (Molecular Probes, Grand Island, NY).

Immunoprecipitation:

Article Title: Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/β-Catenin Signaling Pathway in Glioma Cells
Article Snippet: Western Blot Analysis Cells were lysed with immunoprecipitation assay lysis buffer (Protech Inc., Taipei, Taiwan), and the protein concentration was quantified using a protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The following primary antibodies were used: phospho-AMPK (Thr-172; 1:1000; 2535S), AMPK (1:1000; 2532S), c-caspase-3 (1:1000; 9664S), caspase-3 (1:1000; 9662S), Beclin1 (1:1000; 3495S), Bax (1:1000; 2772S), p21(1:1000; 2947S), Bcl-xL (1:1000; 2764S), and phospho-P53 (p15; 1:1000; 9284S), obtained from Cell Signaling.

Protease Inhibitor:

Article Title: Up-Regulation of MicroRNA-21 Correlates with Lower Kidney Cancer Survival
Article Snippet: Western analysis Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). .. All antibodies, which included p21(cat. no. 2552), p38 MAPkinase (cat. no. 9212) and cyclinE2 (cat. no. 4132) were purchased from Cell Signaling (Danvers, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Culture:

Article Title: Targeting Polo-like kinase 1 in SMARCB1 deleted atypical teratoid rhabdoid tumor
Article Snippet: Antibodies used for western blot analysis were from the following sources: actin #8H10D10, p21#2947, Rad 51#8875, 53BP1#4937, Plk1#5472, ki67# 9449, KU80#2753, Phospho-H2AX#2577 were purchased from Cell Signaling, USA; p53 ab179477 from abcam, INI-1(Anti-BAF47) from BD Biosciences, USA. .. The short-term ATRT cell culture (MAF-A737) was established from a surgical sample obtained from Children’s Hospital Colorado under an approved Institutional Review Board [ ] protocol.

Imaging:

Article Title: A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region
Article Snippet: The protein bands were visualized using chemiluminescence substrate, and images were acquired using a Tanon-4200SF gel imaging system (Shanghai, China). .. Antibodies to β-actin (#4970, Cell Signaling Technology, MA, USA), Phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), Phospho-p53 (Ser15) (#9286, Cell Signaling Technology), γH2AX (#9718, Cell Signaling Technology), C-MYC (#9402, Cell Signaling Technology), P21(#2947, Cell Signaling Technology), P27(#3686, Cell Signaling Technology), anti-rabbit IgG-HRP (#7074, Cell Signaling Technology), and anti-mouse IgG-HRP (#7076, Cell Signaling Technology) were used.

Protein Concentration:

Article Title: Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/β-Catenin Signaling Pathway in Glioma Cells
Article Snippet: Western Blot Analysis Cells were lysed with immunoprecipitation assay lysis buffer (Protech Inc., Taipei, Taiwan), and the protein concentration was quantified using a protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The following primary antibodies were used: phospho-AMPK (Thr-172; 1:1000; 2535S), AMPK (1:1000; 2532S), c-caspase-3 (1:1000; 9664S), caspase-3 (1:1000; 9662S), Beclin1 (1:1000; 3495S), Bax (1:1000; 2772S), p21(1:1000; 2947S), Bcl-xL (1:1000; 2764S), and phospho-P53 (p15; 1:1000; 9284S), obtained from Cell Signaling.

Binding Assay:

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: Non-specific binding was blocked by incubation of the membranes in TBST buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% Tween-20) containing 5% non-fat milk. .. Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling).

Nucleic Acid Electrophoresis:

Article Title: Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/β-Catenin Signaling Pathway in Glioma Cells
Article Snippet: Each lane containing 20 µg of total protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane. .. The following primary antibodies were used: phospho-AMPK (Thr-172; 1:1000; 2535S), AMPK (1:1000; 2532S), c-caspase-3 (1:1000; 9664S), caspase-3 (1:1000; 9662S), Beclin1 (1:1000; 3495S), Bax (1:1000; 2772S), p21(1:1000; 2947S), Bcl-xL (1:1000; 2764S), and phospho-P53 (p15; 1:1000; 9284S), obtained from Cell Signaling.

Immunohistochemistry:

Article Title: Combined autophagy and HDAC inhibition
Article Snippet: Paragraph title: Immunohistochemistry ... Slides were exposed to MAP1LC3B (Abcam, AB48394), CDKN1A (Cell Signaling Technologies, 2947), SQSTM1 (Abcam, AB91526), MKI67 (Cell Signaling Technologies, 9027), active CASP3 (Cell Signaling Technologies, 9661) and CTSD (Abcam, AB91526) antibodies diluted in the protein block solution at 4 °C overnight as previously described.

Article Title: SOX2 suppresses CDKN1A to sustain growth of lung squamous cell carcinoma
Article Snippet: Paragraph title: Immunohistochemistry ... A primary antibody specific for human SOX2 and CDKN1A was obtained from Cell Signaling Technology (Beverly, MA).

Labeling:

Article Title: SOX2 suppresses CDKN1A to sustain growth of lung squamous cell carcinoma
Article Snippet: A primary antibody specific for human SOX2 and CDKN1A was obtained from Cell Signaling Technology (Beverly, MA). .. The slides were treated with streptavidin-biotin complex (Envision System labeled polymer, horseradish peroxidase [HRP], Dako, Carpinteria, CA) for 60 minutes at room temperature.

Polyacrylamide Gel Electrophoresis:

Article Title: Network Pharmacology Integrated Molecular Docking Reveals the Antiosteosarcoma Mechanism of Biochanin A
Article Snippet: Protein samples (100 μ l) from each group were obtained and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on to polyvinylidene fluoride membrane (PVDF). .. Each group of membrane was subsequently treated with dilutions of primary antibody specific for BGLAP (Abcam, Cambridge, USA), ATF3 (Abcam, Cambridge, USA), BAX (Cell Signaling Technology, Danvers, USA), CDKN1A (Cell Signaling Technology, Danvers, USA), and TP53 (Cell Signaling Technology, Danvers, USA) or endogenous reference GAPDH and incubated at 4°C overnight.

SDS Page:

Article Title: Baicalein inhibits pancreatic cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream Akt and ERK signaling pathways
Article Snippet: Protein samples (20 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. .. Primary antibodies used: NEDD9 (#4044, 1:500), Akt (#4691, 1:1000), p-Akt S-473 (#4060, 1:1000), p-Akt T-308 (#2965, 1:1000), ERK1/2 (#9102, 1:1000), p-ERK1/2 (#9101, 1:1000), PDK1(#3062, 1:1000), P21(#8831, 1:1000), P27(#3686, 1:1000) and cleaved caspase-9 (#7237, 1:1000) from Cell Signaling Technology Inc. (Danvers, MA, USA), Bax (AB026, 1:1000) and Bcl-2 (AB112, 1:1000) from Beyotime Biotechnology (Shanghai, China).

Article Title: A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region
Article Snippet: In total, 60 μg of protein was resolved on a 12% SDS-PAGE and was transferred to 0.22-μm immobilon polyvinyl difluoride (PVDF) membranes. .. Antibodies to β-actin (#4970, Cell Signaling Technology, MA, USA), Phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), Phospho-p53 (Ser15) (#9286, Cell Signaling Technology), γH2AX (#9718, Cell Signaling Technology), C-MYC (#9402, Cell Signaling Technology), P21(#2947, Cell Signaling Technology), P27(#3686, Cell Signaling Technology), anti-rabbit IgG-HRP (#7074, Cell Signaling Technology), and anti-mouse IgG-HRP (#7076, Cell Signaling Technology) were used.

Article Title: Network Pharmacology Integrated Molecular Docking Reveals the Antiosteosarcoma Mechanism of Biochanin A
Article Snippet: Protein samples (100 μ l) from each group were obtained and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on to polyvinylidene fluoride membrane (PVDF). .. Each group of membrane was subsequently treated with dilutions of primary antibody specific for BGLAP (Abcam, Cambridge, USA), ATF3 (Abcam, Cambridge, USA), BAX (Cell Signaling Technology, Danvers, USA), CDKN1A (Cell Signaling Technology, Danvers, USA), and TP53 (Cell Signaling Technology, Danvers, USA) or endogenous reference GAPDH and incubated at 4°C overnight.

Software:

Article Title: L-Leucine improves the anaemia in models of Diamond Blackfan anaemia and the 5q-syndrome in a TP53-independent way
Article Snippet: Cells were incubated with primary antibody for CDKN1A (#12D1; Cell Signaling Technology, Beverly, MA) followed by anti-rabbit antibody AlexaFluor 750 (Molecular Probes, Grand Island, NY). .. Flow cytometry was performed on a FACSCanto cytometer (BD Biosciences, San Jose, CA) and data were analysed using FlowJo software version 8.7.1 (TreeStar, Ashland, OR).

Article Title: Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells
Article Snippet: Blots were incubated with rabbit antibodies raised against AR (06–680, EMD Millipore, Billerica, MA), β-Actin (4970, Cell Signaling, Boston, MA), CCND1 (2978, Cell Signaling), CCNE2 (4132, Cell Signaling), phospho-CDK2 (Thr160, 2561, Cell Signaling), CDK2 (2546, Cell Signaling), CDK4 (12790, Cell Signaling), CDKN1A (2947, Cell Signaling), GAPDH (2118, Cell Signaling) or mouse antibodies raised against CCND2 (ab3085, Abcam, Cambridge, MA), CCNA1 (MAB7046, R & D Systems, Minneapolis, MN), CDK4 (2906, Cell Signaling), CDK6 (3136, Cell Signaling), phospho-RB (Ser780, 9307, Cell Signaling), phospho-RB (Ser807/811, 9308, Cell Signaling), or total RB (9309, Cell Signaling), followed by incubation with anti-rabbit or anti-mouse IgG, horseradish peroxidase (HRP)-linked antibody (7074 and #7076, Cell Signaling). .. Films were scanned and Image J software was used to quantify protein expression relative to GAPDH, β-Actin, or total RB controls.

Radio Immunoprecipitation:

Article Title: Up-Regulation of MicroRNA-21 Correlates with Lower Kidney Cancer Survival
Article Snippet: Western analysis Whole-cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA; Thermo Scientific, Rockford, IL, USA; 50 mmol l–1 Tris (pH 8.0), 150 mmol l–1 NaCl, 0.5% deoxycholate, 0.1% SDS and 1.0% NP-40) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). .. All antibodies, which included p21(cat. no. 2552), p38 MAPkinase (cat. no. 9212) and cyclinE2 (cat. no. 4132) were purchased from Cell Signaling (Danvers, MA, USA), whereas GAPDH (cat. no. sc-32233) was from Santa Cruz Biotechnology (Santa Cruz, CA).

Concentration Assay:

Article Title: Targeting Polo-like kinase 1 in SMARCB1 deleted atypical teratoid rhabdoid tumor
Article Snippet: An equivalent amount of DMSO for the highest concentration of drug was used for each experiment as a vehicle control. .. Antibodies used for western blot analysis were from the following sources: actin #8H10D10, p21#2947, Rad 51#8875, 53BP1#4937, Plk1#5472, ki67# 9449, KU80#2753, Phospho-H2AX#2577 were purchased from Cell Signaling, USA; p53 ab179477 from abcam, INI-1(Anti-BAF47) from BD Biosciences, USA.

Article Title: Network Pharmacology Integrated Molecular Docking Reveals the Antiosteosarcoma Mechanism of Biochanin A
Article Snippet: Western-Blot Assay Total protein from each group was extracted and the concentration was determined using concentration Bradford reagent. .. Each group of membrane was subsequently treated with dilutions of primary antibody specific for BGLAP (Abcam, Cambridge, USA), ATF3 (Abcam, Cambridge, USA), BAX (Cell Signaling Technology, Danvers, USA), CDKN1A (Cell Signaling Technology, Danvers, USA), and TP53 (Cell Signaling Technology, Danvers, USA) or endogenous reference GAPDH and incubated at 4°C overnight.

Lysis:

Article Title: Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/β-Catenin Signaling Pathway in Glioma Cells
Article Snippet: Western Blot Analysis Cells were lysed with immunoprecipitation assay lysis buffer (Protech Inc., Taipei, Taiwan), and the protein concentration was quantified using a protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA). .. The following primary antibodies were used: phospho-AMPK (Thr-172; 1:1000; 2535S), AMPK (1:1000; 2532S), c-caspase-3 (1:1000; 9664S), caspase-3 (1:1000; 9662S), Beclin1 (1:1000; 3495S), Bax (1:1000; 2772S), p21(1:1000; 2947S), Bcl-xL (1:1000; 2764S), and phospho-P53 (p15; 1:1000; 9284S), obtained from Cell Signaling.

Article Title: A newly identified berberine derivative induces cancer cell senescence by stabilizing endogenous G-quadruplexes and sparking a DNA damage response at the telomere region
Article Snippet: Western blotting The cells treated with compounds or control medium were collected and lysed in RIPA lysis buffer (Bioteke, China), and the protein concentrations were determined using a BCA protein assay kit (Pierce, U.S.A.). .. Antibodies to β-actin (#4970, Cell Signaling Technology, MA, USA), Phospho-ATM (Ser1981) (#5883, Cell Signaling Technology), Phospho-p53 (Ser15) (#9286, Cell Signaling Technology), γH2AX (#9718, Cell Signaling Technology), C-MYC (#9402, Cell Signaling Technology), P21(#2947, Cell Signaling Technology), P27(#3686, Cell Signaling Technology), anti-rabbit IgG-HRP (#7074, Cell Signaling Technology), and anti-mouse IgG-HRP (#7076, Cell Signaling Technology) were used.

Article Title: Evaluation of the antitumor effects of c-Myc-Max heterodimerization inhibitor 100258-F4 in ovarian cancer cells
Article Snippet: Western blot analysis Total proteins from SKOV3 and Hey cells were extracted in lysis buffer (Thermo Fisher Scientific, Rockford, IL) and quantified using the BCA method. .. After transfer in cold room, the polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) were incubated overnight at 4°C with the following antibodies: beta-actin(1:3000; Santa Cruz Biotechnology, Santa Cruz, CA), cyclin D1(1:1000), CDK4(1:1000), CDK6(1:1000), p27 (1:1000) and p21(1:1000) (Cell Signaling, Boston, MA, USA).

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  • 99
    Cell Signaling Technology Inc p21
    Losmapimod inhibits gefitinib-induced tetraploidization in gefitinib-resistant NSCLC cells. (A) Cell cycle analysis in untreated gefitinib-resistant NSCLC cells (left, HCC827GR and right, H1975) and in cells treated for 24 h with gefitinib or a p38 MAPK inhibitor (SB203580 or losmapimod) either alone or in combination. Tetraploid subpopulations were analyzed by the ModFit LT V4.0 software program. (B) Western blot analysis of p-YAP, p-MKK3/6 and p-p38 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 2, 6 or 10 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total YAP, MKK3/6 and p38 MAPK proteins were used as loading controls. (C) Western blot analysis of p-STAT3, <t>p21</t> and cyclin D1 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 10 or 24 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total STAT3 and GAPDH proteins were used as loading controls.
    P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 681 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p21/product/Cell Signaling Technology Inc
    Average 99 stars, based on 681 article reviews
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    p21 - by Bioz Stars, 2020-02
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    99
    Cell Signaling Technology Inc anti p21
    NCL-6/S*A expression results in an increased expression of apoptotic markers downstream the p53 pathway. (A) Western blot analyses for cells grown without Dx for the indicated period indicating inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A expression resulted in an increase in p53 and <t>p21</t> protein levels. Increased expression of BH3-only pro-apoptotic markers (BID, BIM and PUMA) were observed as early as 1 d or 4 d of induced NCL-6/S*A expression. (B) Plots of p53, p21 and BH3-only protein levels shown in 6A. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 1) cells. The graph is representative of at least two independent experiments.
    Anti P21, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p21/product/Cell Signaling Technology Inc
    Average 99 stars, based on 29 article reviews
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    Losmapimod inhibits gefitinib-induced tetraploidization in gefitinib-resistant NSCLC cells. (A) Cell cycle analysis in untreated gefitinib-resistant NSCLC cells (left, HCC827GR and right, H1975) and in cells treated for 24 h with gefitinib or a p38 MAPK inhibitor (SB203580 or losmapimod) either alone or in combination. Tetraploid subpopulations were analyzed by the ModFit LT V4.0 software program. (B) Western blot analysis of p-YAP, p-MKK3/6 and p-p38 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 2, 6 or 10 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total YAP, MKK3/6 and p38 MAPK proteins were used as loading controls. (C) Western blot analysis of p-STAT3, p21 and cyclin D1 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 10 or 24 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total STAT3 and GAPDH proteins were used as loading controls.

    Journal: EBioMedicine

    Article Title: Losmapimod Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer by Preventing Tetraploidization

    doi: 10.1016/j.ebiom.2018.01.017

    Figure Lengend Snippet: Losmapimod inhibits gefitinib-induced tetraploidization in gefitinib-resistant NSCLC cells. (A) Cell cycle analysis in untreated gefitinib-resistant NSCLC cells (left, HCC827GR and right, H1975) and in cells treated for 24 h with gefitinib or a p38 MAPK inhibitor (SB203580 or losmapimod) either alone or in combination. Tetraploid subpopulations were analyzed by the ModFit LT V4.0 software program. (B) Western blot analysis of p-YAP, p-MKK3/6 and p-p38 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 2, 6 or 10 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total YAP, MKK3/6 and p38 MAPK proteins were used as loading controls. (C) Western blot analysis of p-STAT3, p21 and cyclin D1 expressions in untreated gefitinib-resistant cells (left, HCC827GR and right, H1975) or cells treated for 10 or 24 h with gefitinib (1 μM) or losmapimod (1 μM) either alone or in combination. Total STAT3 and GAPDH proteins were used as loading controls.

    Article Snippet: All other antibodies, including phospho-p38 MAPK (Cell Signaling Technology Cat# 9211, RRID: AB_331641 ), p38 MAPK (Cell Signaling Technology Cat# 9212, RRID: AB_330713 ), p38α MAPK (Cell Signaling Technology Cat# 9218S, RRID: AB_10694846 ), p21 (Cell Signaling Technology Cat# 2947S, RRID: AB_823586 ), cyclin D1 (Cell Signaling Technology Cat# 2922, RRID: AB_2228523 ), p-MKK3 (Ser189)/MKK6 (Ser207) (Cell Signaling Technology Cat# 9236S, RRID: AB_491009 ), MKK3 (Cell Signaling Technology Cat# 8535S, RRID: AB_1122023 ), MKK6 (Cell Signaling Technology Cat# 8550S, RRID: AB_1122022 ), p-Stat3 (Tyr705) (Cell Signaling Technology Cat# 9145, RRID: AB_2491009 ), Stat3 (Cell Signaling Technology Cat# 9139, RRID: AB_331757 ), p-YAP (Ser109) (Cell Signaling Technology Cat# 46931), p-YAP (Ser127) (Cell Signaling Technology Cat# 13008, RRID: AB_2650553 ), YAP (Cell Signaling Technology Cat# 14074, RRID: AB_2650491 ) and GAPDH (Cell Signaling Technology Cat# 2118, RRID: AB_561053 ) were purchased from Cell Signaling Technology (Danvers, MA).

    Techniques: Cell Cycle Assay, Software, Western Blot

    MTA1-induced upregulation of cyclin D1 and downregulation of p21. Notes: SGC7901 ( A ) and BGC823 ( B ) cells were transfected with control (Ctrl) vector and MTA1 expression vector. After 48 hours of transfection, the protein levels of cyclin D1 and p21 were measured by Western blot analysis. MTA1 expression vectors were transiently transfected into SGC7901 ( C ) and BGC823 ( D ) cells, and the mRNA level was estimated by real-time polymerase chain reaction analysis. GAPDH was used as a loading Ctrl. Bars represent the mean ± standard deviation of three independent experiments. * P

    Journal: OncoTargets and therapy

    Article Title: MTA1 promotes proliferation and invasion in human gastric cancer cells

    doi: 10.2147/OTT.S85383

    Figure Lengend Snippet: MTA1-induced upregulation of cyclin D1 and downregulation of p21. Notes: SGC7901 ( A ) and BGC823 ( B ) cells were transfected with control (Ctrl) vector and MTA1 expression vector. After 48 hours of transfection, the protein levels of cyclin D1 and p21 were measured by Western blot analysis. MTA1 expression vectors were transiently transfected into SGC7901 ( C ) and BGC823 ( D ) cells, and the mRNA level was estimated by real-time polymerase chain reaction analysis. GAPDH was used as a loading Ctrl. Bars represent the mean ± standard deviation of three independent experiments. * P

    Article Snippet: The membrane were blocked at room temperature for 1 hour in Tris-buffered saline containing 0.1% Tween-20 (TBST) and 5% fat-free powdered milk, and incubated overnight with specific antibodies: MTA1 (1:500; Santa Cruz Biotechnology Inc., Dallas, TX, USA), cyclin D1, MMP2, MMP9, p21 (1:1,000; Cell Signaling, Boston, MA, USA), fibronectin (EMD Millipore, Billerica, MA, USA), and GAPDH (ShangHai Kangchen, People’s Republic of China) at 4°C.

    Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors (p21, 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.

    Journal: PLoS ONE

    Article Title: Epstein-Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells

    doi: 10.1371/journal.pone.0017809

    Figure Lengend Snippet: Rta-mediated cell cycle arrest precedes the expressions of viral immediate-early genes. (A) Dox-treated 293TetER, EREV8, and ERKV cells cultured for 24 and 48 h were subjected to flow cytometry analysis to quantify the cellular DNA content. The distributions of cells residing in the G2/M, S, G1, and subG1 stages at each time are shown. The results of three independent experiments were similar, and one representative dataset is shown. (B) Comparative expression kinetics (0–48 h) of cell cycle regulators or viral immediate-early proteins in Dox treated 293TetER, EREV8 and ERKV cells. Down-regulation of cell cycle activators (c-Myc, CDK6, CCND2, phosphorylated pRb) and up-regulation of cell cycle inhibitors (p21, 14-3-3σ) are temporally associated with the expression of Rta in all three cell lines. In comparison, EBV BZLF1 and KSHV K-RTA are not significantly augmented until 48 h, a time that alterations of cell cycle gene are nearly completed. α-tubulin served as a loading control.

    Article Snippet: All other antibodies were commercially available: KSHV K8.1 (ABI, Columbia, MD); CDK6, pRb/S807/S811 and p21 (Cell Signaling Technology, Danvers, MA); c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA); 14-3-3σ (GeneTex, Irvine, CA); CCND2 (BD Pharmingen, Franklin Lakes, NJ); α-tubulin (Millipore, Billerica, MA); β-actin and M2-FLAG (Sigma-Aldrich, St. Louis, MO).

    Techniques: Cell Culture, Flow Cytometry, Cytometry, Expressing

    NCL-6/S*A expression results in an increased expression of apoptotic markers downstream the p53 pathway. (A) Western blot analyses for cells grown without Dx for the indicated period indicating inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A expression resulted in an increase in p53 and p21 protein levels. Increased expression of BH3-only pro-apoptotic markers (BID, BIM and PUMA) were observed as early as 1 d or 4 d of induced NCL-6/S*A expression. (B) Plots of p53, p21 and BH3-only protein levels shown in 6A. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 1) cells. The graph is representative of at least two independent experiments.

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: NCL-6/S*A expression results in an increased expression of apoptotic markers downstream the p53 pathway. (A) Western blot analyses for cells grown without Dx for the indicated period indicating inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A expression resulted in an increase in p53 and p21 protein levels. Increased expression of BH3-only pro-apoptotic markers (BID, BIM and PUMA) were observed as early as 1 d or 4 d of induced NCL-6/S*A expression. (B) Plots of p53, p21 and BH3-only protein levels shown in 6A. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 1) cells. The graph is representative of at least two independent experiments.

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Expressing, Western Blot, Software

    NCL-WT and 6/S*A interact with endogenous NCL. Nuclear extracts (NE) were prepared from cells grown without Dx for at least 10 d for NCL-WT or 6/S*A expression. Ctrl represents control cells without exogenous NCL expression. Equal amounts of NE protein from these cells were then subjected to co-immunoprecipitation using anti-FLAG M2 beads. Western analyses of NE and bound fractions were analyzed by anti-NCL (to detect: -exogenous 3xFlag-tagged NCL, upper band and –endogenous NCL, lower band), anti-Flag, anti-p53 and anti-p21. Anti-TOPOII β blot serves as the loading control for NE. The data is representative of three independent experiments performed with 10 d–20 d of WT or 6/S*A expression. This data supports the hypothesis that NCL (both WT and 6/S*A) can associate with endogenous NCL (i.e. NCL-NCL interactions).

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: NCL-WT and 6/S*A interact with endogenous NCL. Nuclear extracts (NE) were prepared from cells grown without Dx for at least 10 d for NCL-WT or 6/S*A expression. Ctrl represents control cells without exogenous NCL expression. Equal amounts of NE protein from these cells were then subjected to co-immunoprecipitation using anti-FLAG M2 beads. Western analyses of NE and bound fractions were analyzed by anti-NCL (to detect: -exogenous 3xFlag-tagged NCL, upper band and –endogenous NCL, lower band), anti-Flag, anti-p53 and anti-p21. Anti-TOPOII β blot serves as the loading control for NE. The data is representative of three independent experiments performed with 10 d–20 d of WT or 6/S*A expression. This data supports the hypothesis that NCL (both WT and 6/S*A) can associate with endogenous NCL (i.e. NCL-NCL interactions).

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Expressing, Immunoprecipitation, Western Blot

    NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression. (A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: NARF6-NCL clones with inducible NCL (WT or 6/S*A) expression. (A) Western blot of a representative clone that expresses 3xFlag-NCL under Tet-off promoter in NARF6 cells (derived from U2OS). (B) Western blot analyses for cells grown without Dx for the indicated period showing inducible NCL-expression (WT or 6/S*A). Both WT and 6/S*A led to a net increase in p53 protein levels and corresponding p21 protein levels -the downstream target of p53. (C) Plots of p53 and p21 protein levels shown in 2B. The quantification was done by NIH Image J software. Values were first corrected for the β-actin levels and then compared to Ctrl (no exogenous NCL, no Dx day 7) cells. The graph is representative of at least three independent experiments.

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Clone Assay, Expressing, Western Blot, Derivative Assay, Software

    Mechanistic model by which nucleolin phosphorylation by CK2 regulates cell proliferation. NCL-WT and phosphorylation-deficient mutant (6/S*A) activate p53 checkpoint and increase corresponding p21 levels. However, NCL-WT expression allows cells through S-phase progression and resumes cell cycle. On the other hand, NCL-6/S*A acts as a dominant-negative mutant that negates NCL-WT functions possibly through oligomerization causing inhibition of cell proliferation and initiating apoptotic pathway.

    Journal: PLoS ONE

    Article Title: Induced Expression of Nucleolin Phosphorylation-Deficient Mutant Confers Dominant-Negative Effect on Cell Proliferation

    doi: 10.1371/journal.pone.0109858

    Figure Lengend Snippet: Mechanistic model by which nucleolin phosphorylation by CK2 regulates cell proliferation. NCL-WT and phosphorylation-deficient mutant (6/S*A) activate p53 checkpoint and increase corresponding p21 levels. However, NCL-WT expression allows cells through S-phase progression and resumes cell cycle. On the other hand, NCL-6/S*A acts as a dominant-negative mutant that negates NCL-WT functions possibly through oligomerization causing inhibition of cell proliferation and initiating apoptotic pathway.

    Article Snippet: Pro-apoptotic Bcl2 family antibody kit and anti-p21, mouse monoclonal were purchased from Cell Signaling technology.

    Techniques: Mutagenesis, Expressing, Dominant Negative Mutation, Inhibition