p73 ab2  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: ( A ) Analysis of TP73 , c-MYC and MYCN genes transcript levels in a panel of human neuroblastoma (NB) cell lines. Semi-quantitative reverse transcription-polymerase chain reaction (RT–PCR) was used to monitor the levels of p73α and p73β transcripts. TAp73 , Δex2-3p73 , ΔNp73 , c-MYC and MYCN transcript levels were evaluated using real-time quantitative RT–PCR (qRT–PCR). ( B ) Western blot analysis of p73, MYCN and p53 protein levels and status (isoform, mutation) in human NB cell lines. For p53, WT refers to the wild-type protein; dupl. ex 7–9 and Δ 373–393 correspond to mutations with duplication of exons 7–9 and deletion of aminoacids 327–393, respectively; the β isoform is a recently described p53 variant generated by alternative splicing in a cryptic site causing a premature stop codon in the C-terminal region. ( C ) Table summarizing the expression and isoform/mutational status of p73, MYCN and p53 protein in this series of human NB cell lines.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described in one of our recent reports , using 4 μg of mouse monoclonal antibody raised against p73 (Ab2, Calbiochem).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Mutagenesis, Variant Assay, Generated, Alternative Splicing, Expressing

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: P73 inhibits MYCN expression at the protein and RNA levels in human neuroblastoma cells. ( A ) Effects of ectopic expression of p73 isoforms (top panel) on MYCN protein (top panel) and MYCN RNA (bottom panel) levels in IMR32 neuroblastoma (NB) cells. Protein levels were monitored by western blotting (top panel). As the first p73 antibody used preferentially recognizes the α isoform, western blot analysis was also performed with an anti-p73β antibody. Ectopic expression of TAp73β also resulted in a moderate up-regulation of p73α, likely as a consequence of transactivation of genes such as that encoding E2F1 that is known to activate TAp73α expression. MYCN transcript levels were evaluated in matching RNA samples using real-time quantitative RT–PCR (qRT–PCR, bottom panel). ( B ) Similar analyses were performed to evaluate the impact of small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 on MYCN protein (top panel) and MYCN RNA (bottom panel) levels in Kelly and LAN-1 NB cells. Plotted qRT–PCR values are the means ± SEM of three replicates.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described in one of our recent reports , using 4 μg of mouse monoclonal antibody raised against p73 (Ab2, Calbiochem).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Small Interfering RNA

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: P73α but not β isoforms lead to activation of MYCN gene transcription in human neuroblastoma cells. Effects of ectopic expression of p73 isoforms ( A and C ) or small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 ( B and D ) on MYCN gene promoter activity in neuroblastoma cell lines. Values represent the mean Luciferase reporter gene activity ± SEM of three replicates.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described in one of our recent reports , using 4 μg of mouse monoclonal antibody raised against p73 (Ab2, Calbiochem).

Techniques: Activation Assay, Expressing, Small Interfering RNA, Activity Assay, Luciferase

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: The p73 protein interacts with MYCN mRNA. Binding of the p73 protein to MYCN transcript was tested by RNA immunoprecipitation (RNA IP) in LAN-1 cells. RNA samples were purified from non-precipitated cellular lysates (input) or extracts precipitated with an antibody raised against p73 (anti-p73 Ab), or a pre-immune serum (Control Ab). Immunoprecipitated MYCN transcripts were detected using real-time quantitative reverse transcription-polymerase chain reaction (qRT–PCR). cDNA: RNA subjected to reverse transcription; RT(−): samples in which reverse transcriptase was omitted from the reaction, used as negative controls. Plotted qRT–PCR values (ratios of RNA IP/input) are the means ± SEM of three replicates.

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described in one of our recent reports , using 4 μg of mouse monoclonal antibody raised against p73 (Ab2, Calbiochem).

Techniques: Binding Assay, RNA Immunoprecipitation, Purification, Control, Immunoprecipitation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: TAp73α inhibits MYCN mRNA stability in human neuroblastoma cells. Effects of ectopic expression of TAp73α ( A ) or small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 ( B and C ) on MYCN mRNA stability in neuroblastoma (NB) cell lines. NB cells were transfected with the indicated control or p73 isoforms expression vectors (A) or siRNA (B, C), then treated with Actinomycin D (5 µM) to block transcription, and collected at different time points for RNA analysis. Logarithms of MYCN mRNA levels (determined by real-time quantitative reverse transcription-polymerase chain reaction—qRT–PCR—. Plotted values are the means ± standard error of the mean—SEM—of three replicates) are displayed as a function of time. The slope of the straight lines obtained in the different experimental conditions (top panels) allowed us to calculate the half-life of MYCN mRNA in response to p73 levels engineering in the three studied cell lines (bottom panels).

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described in one of our recent reports , using 4 μg of mouse monoclonal antibody raised against p73 (Ab2, Calbiochem).

Techniques: Expressing, Small Interfering RNA, Transfection, Control, Blocking Assay, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: p73α isoforms drive opposite transcriptional and post-transcriptional regulation of MYCN expression in neuroblastoma cells

doi: 10.1093/nar/gkn394

Figure Lengend Snippet: TAp73 exerts different effects on the regulation of MYCN and c-MYC expression. Effect of small interfering RNA (siRNA)-mediated depletion of endogenous TAp73 on MYCN and c-MYC mRNA ( A ) and c-MYC protein levels ( B ) in SH-SY5Y neuroblastoma (NB) cells. RNA levels were evaluated using real-time quantitative reverse transcription-polymerase chain reaction (qRT–PCR, A). Plotted qRT–PCR values are the means ± SEM of three replicates. Protein levels were monitored by western blotting (B). Transfection with TAp73 siRNA led to inhibition of both TA- and ΔN-isoforms of p73, as expected from the fact that TAp73 is known to positively regulate the expression of the ΔNp73 isoform. Control and TAp73 siRNA-transfected SH-SY5Y cells were treated with Actinomycin D (5 µM) to block transcription and collected at different time points for RNA analysis. Logarithms of MYCN and c-MYC mRNA levels (plotted values being the means ± SEM of three replicates) are displayed as a function of time. The slope of the straight lines obtained ( C ) allowed us to calculate the half-life of MYCN and c-MYC transcripts in response to p73 depletion in the SH SY5Y cells ( D ).

Article Snippet: Chromatin immunoprecipitation (ChIP) was performed as described in one of our recent reports , using 4 μg of mouse monoclonal antibody raised against p73 (Ab2, Calbiochem).

Techniques: Expressing, Small Interfering RNA, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Transfection, Inhibition, Control, Blocking Assay