rabbit polyclonal anti cyclin d1 m 20 antibody  (Santa Cruz Biotechnology)

Journal: Tumor Biology

Article Title: Identification of interleukin-16 production on tumor aggravation in hepatocellular carcinoma by a proteomics approach

doi: 10.3233/tub-211507

Figure Lengend Snippet: Fig. 4. Western blot band intensities of (a) ERK1/2 protein and phosphorylated ERK1/2 protein and (b) cyclin D1 in HCC cell lines. HCC cells were treated with IL-16 for 48 h. Total ERK1/2 increased significantly in HepG2 and Huh7 cells in a dose-dependent manner. Data indicate the mean ± SEM. ERK1/2 and phosphorylated ERK1/2 data are representative of six independent experiments. Cyclin D1 data are representative of five (HepG2) and six (Huh7) independent experiments. -Tubulin was used as a loading control and to normalize protein intensities. ERK, extracellular signal-regulated protein kinase.

Article Snippet: In addition to anti-human IL-16 Ab, the following Abs were used: rabbit anti-ERK1/2 polyclonal Ab, rabbit anti-phospho-ERK1/2 polyclonal Ab, and rabbit anti-cyclin D1 polyclonal Ab (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27 KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

doi: 10.33594/000000308

Figure Lengend Snippet: Fig. 5. Delayed G1/S cell cycle transition is accompanied by up-regulation of p27KIP1 in myoblasts depleted of CK2α and in CK2α-KO mouse embryos. (A) Cells were synchronized by serum starvation in the presence or absence of doxycycline for 48 hours and harvested at the indicated time points after adding full growth medium. (B) Total RNA was isolated and used for reverse transcription quantitative PCR (RT-qPCR). Graph shows the ratio p27KIP1/HPRT1 mRNA. Data are the average of three independent experiments +/- standard error of the mean (SEM). *P = 0.02, **P = 0.0031, ***P = 0.004. (C) Whole lysate from cells treated as described in (A) were analyzed by western blot employing antibodies against the indicated proteins. (D) Immunostaining of cells with rabbit polyclonal anti-p27KIP1 and subsequently, a biotin-conjugated secondary anti-rabbit IgG antibody. Expression and localization of p27KIP1 was revealed by cell staining with Alexa Fluor 555-conjugated streptavidin. Cell nuclei were visualized by DAPI staining. Cell pictures were taken at 40x magnification. (E) p27KIP1 staining of tissue sections from wild-type (+/+) and knockout (-/-) mouse embryos at E10.5, respectively. Photographs were taken at 20x magnification. Fluorescence pictures were pseudo-colored and show p27KIP1 nuclear staining (red). Cell nuclei were visualized as in (D). (F) Bar-graph showing in percentage the ratio of p27KIP1-positive cells/total number of cells in the embryos. Three to five sections each from three pairs of wild-type and knockout embryos were analyzed. Values are mean +/- STDEV, *P<0.0005. SO: somite.

Article Snippet: Proteins were detected by incubating western blot membranes with the following antibodies: polyclonal anti-CK2α obtained by immunizing rabbits against the human full-length protein sequence; polyclonal anti-CK2α’ obtained by immunizing rabbits with a specific peptide sequence of human CK2α’ (i.e. SQPCADNAVLSSGTAAR); mouse monoclonal anti-CK2b (KinaseDetect Aps, Odense, Denmark); rabbit polyclonal anti-phospho-Rb (S795), rabbit monoclonal anti-cyclin E1, rabbit monoclonal anti-p27KIP1, rabbit polyclonal anti-AKT, rabbit polyclonal anti-phospho-AKT (T308), rabbit polyclonal anti-acetyl CoA carboxylase (ACC), rabbit polyclonal anti-phospho-ACC (S79), rabbit polyclonal anti-AMPKα, rabbit monoclonal anti-FoxO1, rabbit monoclonal anti-FoxO3A, rabbit monoclonal anti-phospho-FoxO3A (S253), rabbit monoclonal anti-MCM4 and rabbit monoclonal anti-MCM7 (all from Cell Signaling Technology, MA, USA); mouse monoclonal anti-Rb, rabbit polyclonal anti-cyclin A, rabbit polyclonal anti-cyclin D1, rabbit polyclonal anti-E2F, goat polyclonal anti-MCM3, goat polyclonal anti-MCM6, mouse monoclonal anti-p21WAF1 and mouse monoclonal anti-FoxO3A (all from Santa Cruz Biotechnology, Heidelberg, Germany); rabbit monoclonal anti-phopho-p27KIP1 (S10, Abcam, Cambridge, United Kingdom); mouse monoclonal anti-βactin (Sigma-Aldrich); rabbit polyclonal anti-phospho-p27KIP1 (T197) (R&D Systems, Abingdon, UK); rabbit polyclonal anti-phospho-AKT (S129) (Abgent, San Diego, CA, USA).

Techniques: Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunostaining, Expressing, Staining, Knock-Out, Fluorescence

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27 KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells.

doi: 10.33594/000000308

Figure Lengend Snippet: Fig. 6. Up-regulation of p27KIP1 correlates with slightly higher levels of FoxO3A in cells with lowered expression of CK2α. However, FoxO3A silencing does not affect the expression of p27KIP1. (A) Cells were serum-starved for 48 hours in the presence or absence of doxycycline and harvested at the indicated time points. Whole cell lysate was analyzed by western blot employing antibodies against FoxO1 and FoxO3A, respectively. Detection of β-actin confirmed equal protein loading. (B) Cells treated as indicated in (A) were labeled with rabbit polyclonal anti- FoxO3A. Proteins were visualized by staining with Alexa Fluor 555-conjugated streptavidin (red fluorescence) after incubation with a secondary anti-rabbit IgG antibody conjugated with biotin. Cellular DNA was detected by staining with DAPI reagent (blue fluorescence). Cell pictures were taken at 40x magnification. (C) Cells were rendered quiescent by serum withdrawal. Subsequently, they were stimulated by adding 100 ng/ml IGF-I and harvested at the indicated time points as shown in the Fig.. Whole cell extract was analyzed by western blot employing antibodies against the indicated proteins. (D) Down-regulation of FoxO3A was obtained by transfecting cells with siRNA targeting FoxO3A-mRNA. Control cells were transfected with scrambled siRNA. Cells were synchronized by serum starvation and harvested after 0 hours (i.e. corresponding to quiescent cells) and 6 hours after stimulation. Experiments were repeated at least three times obtaining similar results. One representative experiment is shown.

Article Snippet: Proteins were detected by incubating western blot membranes with the following antibodies: polyclonal anti-CK2α obtained by immunizing rabbits against the human full-length protein sequence; polyclonal anti-CK2α’ obtained by immunizing rabbits with a specific peptide sequence of human CK2α’ (i.e. SQPCADNAVLSSGTAAR); mouse monoclonal anti-CK2b (KinaseDetect Aps, Odense, Denmark); rabbit polyclonal anti-phospho-Rb (S795), rabbit monoclonal anti-cyclin E1, rabbit monoclonal anti-p27KIP1, rabbit polyclonal anti-AKT, rabbit polyclonal anti-phospho-AKT (T308), rabbit polyclonal anti-acetyl CoA carboxylase (ACC), rabbit polyclonal anti-phospho-ACC (S79), rabbit polyclonal anti-AMPKα, rabbit monoclonal anti-FoxO1, rabbit monoclonal anti-FoxO3A, rabbit monoclonal anti-phospho-FoxO3A (S253), rabbit monoclonal anti-MCM4 and rabbit monoclonal anti-MCM7 (all from Cell Signaling Technology, MA, USA); mouse monoclonal anti-Rb, rabbit polyclonal anti-cyclin A, rabbit polyclonal anti-cyclin D1, rabbit polyclonal anti-E2F, goat polyclonal anti-MCM3, goat polyclonal anti-MCM6, mouse monoclonal anti-p21WAF1 and mouse monoclonal anti-FoxO3A (all from Santa Cruz Biotechnology, Heidelberg, Germany); rabbit monoclonal anti-phopho-p27KIP1 (S10, Abcam, Cambridge, United Kingdom); mouse monoclonal anti-βactin (Sigma-Aldrich); rabbit polyclonal anti-phospho-p27KIP1 (T197) (R&D Systems, Abingdon, UK); rabbit polyclonal anti-phospho-AKT (S129) (Abgent, San Diego, CA, USA).

Techniques: Expressing, Western Blot, Labeling, Staining, Fluorescence, Incubation, Control, Transfection

Journal: Epigenetics

Article Title: PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

doi: 10.1080/15592294.2019.1676597

Figure Lengend Snippet: Figure 2. (a) Methylation of PTPRT promoter region in NSCLC tumours. NSCLC tumours samples from Stage 1A/1B patients were processed to extract DNA followed by bisulphite conversion to determine PTPRT promoter methylation by Real Time PCR. Out of the 12 tumour samples, six tumours had detectable promoter region methylation (Patient #s 2, 4, 6, 8, 9 and 11) whereas the remaining 6 tumours demonstrated very low methylation of the PTPRT promoter region (Patient #s 1, 3, 5, 7, 10 and 12), which compared well with the methylated beta values. (b&c) Association of PTPRT promoter methylation to STAT3 target gene expression in lung tumours. Lung tumours (Patient #s 1, 2 and 3) were assessed for STAT3 target gene expression by RT-PCR. Cyclin D1 (Figure 2(b)) and Bcl-XL (Figure 2(c)) mRNA expression was found to associate with PTPRT methylation status. (d&e) Association of PTPRT promoter methylation and Bcl-XL and Cyclin D1 mRNA expression. Scatter plots of DNA promoter methylation of PTPRT and Bcl-XL and Cyclin D1 mRNA expression in TCGA adenocarcinoma showed an increase in Bcl-XL (Figure 2(d)) and Cyclin D1 (Figure 2(e)) mRNA expression with increase in PTPRT methylation.

Article Snippet: To determine pSTAT3Tyr705, STAT3 and STAT3 targets such as Cyclin D1 and Bcl-XL protein expressions, 40 μg/lane were separated by 10% SDSPAGE and probed with rabbit anti-pSTAT3Tyr705 or mouse anti-STAT3 monoclonal antibodies (Cell Signalling Technology, Boston, MA), rabbit anti- human Cyclin D1 polyclonal or mouse anti-human Bcl-XL monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Massachusetts, MA, USA).

Techniques: Methylation, Real-time Polymerase Chain Reaction, Targeted Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Epigenetics

Article Title: PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

doi: 10.1080/15592294.2019.1676597

Figure Lengend Snippet: Figure 4. (a) Knockdown of PTPRT mRNA expression in H520 cells by PTPRT siRNA. PTPRT knockdown by siRNA in H520 cells which demonstrated PTPRT mRNA expression in our study resulted in decreased expression of PTPRT mRNA. H520 cells were treated with PTPRT siRNA in presence of OPTI-MEM and after 48h, cells were replaced with complete media and at the end of 72h, RNA was extracted. RT-PCR data showed that H520 cells transfected with PTPRT siRNA had significantly lower 48% (p < 0.05) PTPRT mRNA expression compared to cells treated with vehicle. PRPRT mRNA expression in H520 cells transfected with scrambled siRNA did not show any significant decrease when compared to cells treated with vehicle. Results are from three triplicate wells in one experiment. (b & c) Increased expression of Cyclin D1 and Bcl-XL upon PTPRT knockdown in H520 cells. H520 cells which demonstrated unmethylation of the PTPRT promoter region were transfected with PTPRT siRNA. After 72h, the cells showed increased mRNA expression of the STAT3 target genes such as Cyclin D1 and Bcl-XL compared to the cells treated with vehicle or scrambled siRNA as determined by RT-PCR. Results are from three triplicate wells in one experiment. (d & e) Increased pSTAT3Tyr705 along with STAT3 target gene expression upon PTPRT knockdown by siRNA. H520 cells transfected with PTPRT siRNA caused increased pSTAT3Tyr705 along with Cyclin D1 and Bcl-XL protein expression compared to the cells treated with vehicle or scrambled siRNA at the end of 72h and 96h as determined by western analyses. The results are from triplicate wells from one experiment.

Article Snippet: To determine pSTAT3Tyr705, STAT3 and STAT3 targets such as Cyclin D1 and Bcl-XL protein expressions, 40 μg/lane were separated by 10% SDSPAGE and probed with rabbit anti-pSTAT3Tyr705 or mouse anti-STAT3 monoclonal antibodies (Cell Signalling Technology, Boston, MA), rabbit anti- human Cyclin D1 polyclonal or mouse anti-human Bcl-XL monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Massachusetts, MA, USA).

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Targeted Gene Expression, Western Blot

Journal: Epigenetics

Article Title: PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

doi: 10.1080/15592294.2019.1676597

Figure Lengend Snippet: Figure 5. (a, b, c & d) Increased PTPRT promoter methylation is associated with increased sensitivity to STAT3 inhibition. Lung cancer cell lines which are methylated (H23 and H1703) or unmethylated (H520) in the PTPRT promoter region were treated with increasing concentrations of the three STAT3 inhibitors SID 864,669, SID 4,248,543 and UPCDC 10,205 in complete media. At the end of 72h, MTT assay was done to determine % kill which increased with increasing concentrations of SID 864,669 (Figure 5(a)), SID 4,248,543 (Figure 5(b)) and UPCDC 10,205 (Figure 5(c)) and was more pronounced in H23 and H1703 which are methylated in the PTPRT promoter region in conjunction with high pSTAT3Tyr705 expression. The inactive compound UPCDC 10,381 did not have an effect in any cell lines tested (Figure 5(d)). Results are from four separate experiments. (e, f & g) Increased PTPRT promoter methylation was accompanied with increased sensitivity to STAT3 inhibition as evident from the reduction of STAT3 target gene expression. H23, H1703 and H520 cells were treated with varying concentrations of SID 864,669 and at the end of 24h, cells were harvested for RNA extraction and RT-PCR was performed to determine the mRNA expression of the STAT3 target genes. The STAT3 inhibitor SID 864,669 caused a reduction in Cyclin D1 and Bcl-XL mRNA expression in H23 (Figure 5(e), * indicates p < 0.05) and H1703 (Figure 5(f), * indicates p < 0.05) cells which showed PTPRT promoter methylation with concomitant expression of pSTAT3Tyr705. In H520 cells, there was no effect of SID 864,669 on the mRNA expression of STAT3 target genes (Figure 5(g)).

Article Snippet: To determine pSTAT3Tyr705, STAT3 and STAT3 targets such as Cyclin D1 and Bcl-XL protein expressions, 40 μg/lane were separated by 10% SDSPAGE and probed with rabbit anti-pSTAT3Tyr705 or mouse anti-STAT3 monoclonal antibodies (Cell Signalling Technology, Boston, MA), rabbit anti- human Cyclin D1 polyclonal or mouse anti-human Bcl-XL monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and developed using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Massachusetts, MA, USA).

Techniques: Methylation, Inhibition, MTT Assay, Expressing, Targeted Gene Expression, RNA Extraction, Reverse Transcription Polymerase Chain Reaction