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Sino Biological erbb2 ha

A A gene-centric heatmap representing copy number abundance (a minimum of 10% frequency in at least one single type of cancer). Color legend represents % frequency. B HEK293 cells were co-transfected with Super Nanoluciferase reporter vectors containing non-ARE control or RPS30-nLuc-ARE reporter together with of control Firefly luciferase vector, for 18 h. Cells were re-seeded into 96-well microplates and were transfected with empty vector control or <t>ERBB2</t> -HA vector for 18 h. Cells were lysed, and luciferase activity was quantitated as the ratio of Nanoluciferase/Firefly luciferase intensity. The screen is at least from two independent experiments with Mean ± SEM of triplicate readings. ANOVA with Dunnett’s multiple comparisons was used to compare the effects of each of the indicated vectors and the empty vector on ARE-reporter readings. ** p < 0.01, *** p < 0.001, **** p < 0.0001. C Cancer-centric heatmap representing clustering of cancer type according to copy number variations.

Erbb2 Ha, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/erbb2+ha/pmc08448767-252-8-12?v=Sino+Biological
Average 90 stars, based on 6 article reviews

erbb2 ha - by Bioz Stars, 2026-07

90/100 stars

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1) Product Images from "Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway"

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

Journal: Oncogenesis

doi: 10.1038/s41389-021-00351-w

Figure Legend Snippet: A A gene-centric heatmap representing copy number abundance (a minimum of 10% frequency in at least one single type of cancer). Color legend represents % frequency. B HEK293 cells were co-transfected with Super Nanoluciferase reporter vectors containing non-ARE control or RPS30-nLuc-ARE reporter together with of control Firefly luciferase vector, for 18 h. Cells were re-seeded into 96-well microplates and were transfected with empty vector control or ERBB2 -HA vector for 18 h. Cells were lysed, and luciferase activity was quantitated as the ratio of Nanoluciferase/Firefly luciferase intensity. The screen is at least from two independent experiments with Mean ± SEM of triplicate readings. ANOVA with Dunnett’s multiple comparisons was used to compare the effects of each of the indicated vectors and the empty vector on ARE-reporter readings. ** p < 0.01, *** p < 0.001, **** p < 0.0001. C Cancer-centric heatmap representing clustering of cancer type according to copy number variations.

Techniques Used: Transfection, Luciferase, Plasmid Preparation, Activity Assay

A The ERBB2-amplification associated expressed genes (≥1.7 fold, Q value <0.00005) were intersected with the AU-rich element database (ARED-Plus). B Functional enrichment of the ERBB2-associated ARE-coding genes. The most significant results with false discovery rate (FDR) P < 0.05 are shown. C Left panel: the mean of mRNA abundance for 160 ARE-coding genes (Fig. data) was compared between TCGA ERBB2-negative and ERBB2-positive patients’ tissue samples. Data are Mean ± SEM. *** p < 0.001 using Student’s t-test with Welch’s correction. Right panel: the interaction between the ARE-mRNA cluster or all mRNAs with ERBB2-positive gene expression data is represented by an odd ratio using Fisher’s exact test (Oncomine software). D Total RNA from the ERBB2-positive SKBR3 cell line and MCF10A normal-like ERBB2-negative cell lines were extracted and subjected to RT-QPCR using specific primers to the indicated genes. Inset shows an immunoblot of two for the ERBB2 protein among different types of breast cancer cell lines. E The SKBR3 cells were transfected with 200 nM control siRNA or ERBB2 siRNA for 24 h, followed by RNA extraction and RT-QPCR as indicated. Inset is a representative immunoblot of three, for the ERBB2 protein. The data in D and E are normalized to the housekeeping gene, RPL0. Data are Mean ± SEM (triplicate) from one representative experiment of at least two. ANOVA test demonstrates overall statistical significance. The student’s t-test was used to compare expression data for each gene.

Figure Legend Snippet: A The ERBB2-amplification associated expressed genes (≥1.7 fold, Q value <0.00005) were intersected with the AU-rich element database (ARED-Plus). B Functional enrichment of the ERBB2-associated ARE-coding genes. The most significant results with false discovery rate (FDR) P < 0.05 are shown. C Left panel: the mean of mRNA abundance for 160 ARE-coding genes (Fig. data) was compared between TCGA ERBB2-negative and ERBB2-positive patients’ tissue samples. Data are Mean ± SEM. *** p < 0.001 using Student’s t-test with Welch’s correction. Right panel: the interaction between the ARE-mRNA cluster or all mRNAs with ERBB2-positive gene expression data is represented by an odd ratio using Fisher’s exact test (Oncomine software). D Total RNA from the ERBB2-positive SKBR3 cell line and MCF10A normal-like ERBB2-negative cell lines were extracted and subjected to RT-QPCR using specific primers to the indicated genes. Inset shows an immunoblot of two for the ERBB2 protein among different types of breast cancer cell lines. E The SKBR3 cells were transfected with 200 nM control siRNA or ERBB2 siRNA for 24 h, followed by RNA extraction and RT-QPCR as indicated. Inset is a representative immunoblot of three, for the ERBB2 protein. The data in D and E are normalized to the housekeeping gene, RPL0. Data are Mean ± SEM (triplicate) from one representative experiment of at least two. ANOVA test demonstrates overall statistical significance. The student’s t-test was used to compare expression data for each gene.

Techniques Used: Amplification, Functional Assay, Expressing, Software, Quantitative RT-PCR, Western Blot, Transfection, RNA Extraction

A SKBR3 cells were treated with increasing doses of lapatinib for 24 h, and lysed for immunoblotting with anti-phospho ERBB2 protein and β-actin as a loading control. WB is one representative from three blots. B RT-QPCR expression analysis for the ERBB2-regulated ARE-mRNAs in SKBR3 cells (left panel) or MCF-10A cells ( right panel ) treated with DMSO or Lapatinib (300 nM) for overnight. Data are normalized to the housekeeping gene, RPL0. The normalized values were used to calculate % remaining relative to DMSO control. Statistical analysis was performed using two-tailed Student’s t -test for each gene reduced in expression in comparison to DMSO data. Data are Mean ± SEM (triplicate of one experiment of three). ** P < 0.001, *** P < 0.0001. C RT-QPCR for Nanoluciferase mRNA abundance or Nanoluciferase protein activity ( D ) in SKBR3 cells transfected with nLuc-ARE or control nLuc-non-ARE expression plasmid for overnight, and then treated with DMSO (control) or lapatinib for 6 h or 24 h, respectively. Data are presented as the mean ± SEM ( n = 2 experiments). ANOVA is used to compare overall significance while post hoc sidak’s test compares the DMSO and lapatinib data. *** p < 0.0001. ** p < 0.005, Student’s test. E The mRNA half-life determination in response to lapatinib. The SKBR3 cells were treated with DMSO or lapatinib (300 nM) overnight, then actinomycin D (AcD) (5 μg/ml) was added for the indicated time points. Total RNA was extracted, and cDNA samples were subjected to RT-QPCR using specific primers to the indicated genes. One-phase exponential decay curves for relative mRNA half-life measurements in the two treated groups are shown. Data are normalized to RPL0 transcript abundance, and the normalized values were used to calculate the mRNA half-life as a percentage to time 0. Data are presented as the mean ± SEM ( n = 2 experiments). F , G Immunoblot analysis for CDC6 and NEK2 protein abundance in SKBR3 cells treated as in ( A ). Representative blot each is from at least two experiments is shown.

Figure Legend Snippet: A SKBR3 cells were treated with increasing doses of lapatinib for 24 h, and lysed for immunoblotting with anti-phospho ERBB2 protein and β-actin as a loading control. WB is one representative from three blots. B RT-QPCR expression analysis for the ERBB2-regulated ARE-mRNAs in SKBR3 cells (left panel) or MCF-10A cells ( right panel ) treated with DMSO or Lapatinib (300 nM) for overnight. Data are normalized to the housekeeping gene, RPL0. The normalized values were used to calculate % remaining relative to DMSO control. Statistical analysis was performed using two-tailed Student’s t -test for each gene reduced in expression in comparison to DMSO data. Data are Mean ± SEM (triplicate of one experiment of three). ** P < 0.001, *** P < 0.0001. C RT-QPCR for Nanoluciferase mRNA abundance or Nanoluciferase protein activity ( D ) in SKBR3 cells transfected with nLuc-ARE or control nLuc-non-ARE expression plasmid for overnight, and then treated with DMSO (control) or lapatinib for 6 h or 24 h, respectively. Data are presented as the mean ± SEM ( n = 2 experiments). ANOVA is used to compare overall significance while post hoc sidak’s test compares the DMSO and lapatinib data. *** p < 0.0001. ** p < 0.005, Student’s test. E The mRNA half-life determination in response to lapatinib. The SKBR3 cells were treated with DMSO or lapatinib (300 nM) overnight, then actinomycin D (AcD) (5 μg/ml) was added for the indicated time points. Total RNA was extracted, and cDNA samples were subjected to RT-QPCR using specific primers to the indicated genes. One-phase exponential decay curves for relative mRNA half-life measurements in the two treated groups are shown. Data are normalized to RPL0 transcript abundance, and the normalized values were used to calculate the mRNA half-life as a percentage to time 0. Data are presented as the mean ± SEM ( n = 2 experiments). F , G Immunoblot analysis for CDC6 and NEK2 protein abundance in SKBR3 cells treated as in ( A ). Representative blot each is from at least two experiments is shown.

Techniques Used: Western Blot, Quantitative RT-PCR, Expressing, Two Tailed Test, Activity Assay, Transfection, Plasmid Preparation

A immunoblot analysis for ZFP36/TTP phosphorylated protein abundance in MCF10A and SKBR3 using specific primers to ZFP36/TTP and β-Actin protein as a loading control. B SKBR3 cells were transfected with an empty vector or ZFP36/TTP expression vector. Left panel: proteins were extracted, and immunoblotting was performed using a ZFP36/TTP antibody. Right panel: protein lysates were first treated with the phosphatase, CIP, or CIP phosStop C before immunoblotting with anti-ZFP36/TTP. The WB shows both phosphorylated and unphosphorylated bands. WB was performed with low exposure to avoid high background and overexposed signal of the transfected TTP (lower panel). D) SKBR3 cells were transfected with 200 nM ERBB2 siRNA or control siRNA for overnight, and ERBB2 and ZFP36/TTP protein abundance were assessed by immunoblotting. A representative blot from at least two experiments is shown. Immunoblot analysis of ERBB2 and ZFP36/TTP protein phosphorylation abundance in MCF10A ( E ) or MCF-7 ( F ) transfected with ERBB2 expression vector or empty vector. Representative blots are from at least three experiments.

Figure Legend Snippet: A immunoblot analysis for ZFP36/TTP phosphorylated protein abundance in MCF10A and SKBR3 using specific primers to ZFP36/TTP and β-Actin protein as a loading control. B SKBR3 cells were transfected with an empty vector or ZFP36/TTP expression vector. Left panel: proteins were extracted, and immunoblotting was performed using a ZFP36/TTP antibody. Right panel: protein lysates were first treated with the phosphatase, CIP, or CIP phosStop C before immunoblotting with anti-ZFP36/TTP. The WB shows both phosphorylated and unphosphorylated bands. WB was performed with low exposure to avoid high background and overexposed signal of the transfected TTP (lower panel). D) SKBR3 cells were transfected with 200 nM ERBB2 siRNA or control siRNA for overnight, and ERBB2 and ZFP36/TTP protein abundance were assessed by immunoblotting. A representative blot from at least two experiments is shown. Immunoblot analysis of ERBB2 and ZFP36/TTP protein phosphorylation abundance in MCF10A ( E ) or MCF-7 ( F ) transfected with ERBB2 expression vector or empty vector. Representative blots are from at least three experiments.

Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing

A Dose-dependent activity of lapatinib on the abundance of phosphorylated ZFP36/TTP in the ERBB2-positive SKBR3 cells. The relative amounts of the proteins from three immunoblots were quantified using Image J software ( left panel ). Fold changes (Mean ± SEM, n = 3) of phosphorylated ZFP36/TTP abundance normalized to β-Actin using Image J program (right panel). B Effect of ERBB2 inhibition on ZFP36/TTP protein stability. SKBR3 cells were treated with lapatinib (300 nM) for 16 h, followed by 10 µg/ml of cycloheximide (CHX) treatment for the indicated durations (left panel). Protein lysates were subjected to immunoblotting with anti-TTP. Right panel: quantification of ZFP36/TTP abundance, normalized to GAPDH, was determined using ImageJ software and the protein half-life was assessed using the Graph Prism. Data are mean +/− SEM of two experiments. C Effect of lapatinib on the abundance of ZFP36/TTP protein in the ERBB2-positive cells, BT-474. WB is a representative of two blots from independent experiments.

Figure Legend Snippet: A Dose-dependent activity of lapatinib on the abundance of phosphorylated ZFP36/TTP in the ERBB2-positive SKBR3 cells. The relative amounts of the proteins from three immunoblots were quantified using Image J software ( left panel ). Fold changes (Mean ± SEM, n = 3) of phosphorylated ZFP36/TTP abundance normalized to β-Actin using Image J program (right panel). B Effect of ERBB2 inhibition on ZFP36/TTP protein stability. SKBR3 cells were treated with lapatinib (300 nM) for 16 h, followed by 10 µg/ml of cycloheximide (CHX) treatment for the indicated durations (left panel). Protein lysates were subjected to immunoblotting with anti-TTP. Right panel: quantification of ZFP36/TTP abundance, normalized to GAPDH, was determined using ImageJ software and the protein half-life was assessed using the Graph Prism. Data are mean +/− SEM of two experiments. C Effect of lapatinib on the abundance of ZFP36/TTP protein in the ERBB2-positive cells, BT-474. WB is a representative of two blots from independent experiments.

Techniques Used: Activity Assay, Western Blot, Software, Inhibition

A Immunoblot analysis of ERBB2, AKT, MEK1/2, and ERK1/2 signaling inhibition after treatment with increasing doses of lapatinib for 24 h. Status of ERBB2 (phospho-tyrosine 1221/1222 and total), AKT (phospho-serine 473 and total pan AKT), ERK1/2 (Phospho-threonine 202/phospho-tyrosine 204 and total), MEK1/2 (Ser217/221), and ZFP36/TTP are shown. A blot from at least two experiments is shown. B Immunoblot analysis for the inhibition of MK2 activation after treatment with lapatinib. SKBR3 cells were treated with increasing doses of lapatinib, as indicated for 24 h. Phospho-threonine MK2 (T222) and total MK2 protein abundance are shown. A representative blot from two experiments is shown. C Immunoblot analysis for ZFP36/TTP phosphorylation in MAPKAPK-2 silenced SKBR3 cells. SKBR3 cells transfected with 100 nM of si Ctrl or si MAPKAPK-2 for 24 h were tested for ZFP36/TTP protein abundance. Total MK2, phosphorylated ZFP36/TTP, and β-actin protein abundances are shown. A representative blot from three experiments is shown. Effect of the MK2 inhibitor PF-3644022 (1 µM, 4 h) on the abundance of phosphorylated ZFP36/TTP in ERBB2-expressing SKBR3 ( D ) or SKBR3 transfected with ZFP36 expression plasmid ( E ). Western blots are from two independent experiments. F Immunoblot analysis of total and phosphorylated ZFP36/TTP, and MK2 in MK2 -knockout MEF cells in the absence or presence of overnight transfected ERBB2.

Figure Legend Snippet: A Immunoblot analysis of ERBB2, AKT, MEK1/2, and ERK1/2 signaling inhibition after treatment with increasing doses of lapatinib for 24 h. Status of ERBB2 (phospho-tyrosine 1221/1222 and total), AKT (phospho-serine 473 and total pan AKT), ERK1/2 (Phospho-threonine 202/phospho-tyrosine 204 and total), MEK1/2 (Ser217/221), and ZFP36/TTP are shown. A blot from at least two experiments is shown. B Immunoblot analysis for the inhibition of MK2 activation after treatment with lapatinib. SKBR3 cells were treated with increasing doses of lapatinib, as indicated for 24 h. Phospho-threonine MK2 (T222) and total MK2 protein abundance are shown. A representative blot from two experiments is shown. C Immunoblot analysis for ZFP36/TTP phosphorylation in MAPKAPK-2 silenced SKBR3 cells. SKBR3 cells transfected with 100 nM of si Ctrl or si MAPKAPK-2 for 24 h were tested for ZFP36/TTP protein abundance. Total MK2, phosphorylated ZFP36/TTP, and β-actin protein abundances are shown. A representative blot from three experiments is shown. Effect of the MK2 inhibitor PF-3644022 (1 µM, 4 h) on the abundance of phosphorylated ZFP36/TTP in ERBB2-expressing SKBR3 ( D ) or SKBR3 transfected with ZFP36 expression plasmid ( E ). Western blots are from two independent experiments. F Immunoblot analysis of total and phosphorylated ZFP36/TTP, and MK2 in MK2 -knockout MEF cells in the absence or presence of overnight transfected ERBB2.

Techniques Used: Western Blot, Inhibition, Activation Assay, Transfection, Expressing, Plasmid Preparation, Knock-Out

A HEK293 cells were co-transfected with ERBB2 -HA along with ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. Proteins were extracted, and immunoblotting was performed using antibodies to ERBB2, ZFP36/TTP, and β-actin. A representative blot from three experiments is shown. B Total RNA samples were extracted and subjected to RT-QPCR using specific primers to the ERBB2-regulated ARE-mRNA s (as in Fig. D, E). Data are Mean ± SEM of average 18SRNA normalized expression of ARE-mRNA abundance remaining from vector control. C , D RT-QPCR using specific primers to the indicated genes RT-QPCR using specific primers to the indicated genes. Data in B to D are Mean ± SEM from an experiment with triplicate reactions of two independent experiments. Two-way ANOVA with Tukey’s multiple tests between the control and treatment test as indicated were used. *** p < 0.001. E Immunoblot analysis for ZFP36/TTP protein in HEK293 cells over-expressing cancer-amplified genes. HEK293 cells were co-transfected with cancer amplified genes and ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. The results of the two experiments are shown.

Figure Legend Snippet: A HEK293 cells were co-transfected with ERBB2 -HA along with ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. Proteins were extracted, and immunoblotting was performed using antibodies to ERBB2, ZFP36/TTP, and β-actin. A representative blot from three experiments is shown. B Total RNA samples were extracted and subjected to RT-QPCR using specific primers to the ERBB2-regulated ARE-mRNA s (as in Fig. D, E). Data are Mean ± SEM of average 18SRNA normalized expression of ARE-mRNA abundance remaining from vector control. C , D RT-QPCR using specific primers to the indicated genes RT-QPCR using specific primers to the indicated genes. Data in B to D are Mean ± SEM from an experiment with triplicate reactions of two independent experiments. Two-way ANOVA with Tukey’s multiple tests between the control and treatment test as indicated were used. *** p < 0.001. E Immunoblot analysis for ZFP36/TTP protein in HEK293 cells over-expressing cancer-amplified genes. HEK293 cells were co-transfected with cancer amplified genes and ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. The results of the two experiments are shown.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Amplification

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Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A A gene-centric heatmap representing copy number abundance (a minimum of 10% frequency in at least one single type of cancer). Color legend represents % frequency. B HEK293 cells were co-transfected with Super Nanoluciferase reporter vectors containing non-ARE control or RPS30-nLuc-ARE reporter together with of control Firefly luciferase vector, for 18 h. Cells were re-seeded into 96-well microplates and were transfected with empty vector control or ERBB2 -HA vector for 18 h. Cells were lysed, and luciferase activity was quantitated as the ratio of Nanoluciferase/Firefly luciferase intensity. The screen is at least from two independent experiments with Mean ± SEM of triplicate readings. ANOVA with Dunnett’s multiple comparisons was used to compare the effects of each of the indicated vectors and the empty vector on ARE-reporter readings. ** p < 0.01, *** p < 0.001, **** p < 0.0001. C Cancer-centric heatmap representing clustering of cancer type according to copy number variations.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A The ERBB2-amplification associated expressed genes (≥1.7 fold, Q value <0.00005) were intersected with the AU-rich element database (ARED-Plus). B Functional enrichment of the ERBB2-associated ARE-coding genes. The most significant results with false discovery rate (FDR) P < 0.05 are shown. C Left panel: the mean of mRNA abundance for 160 ARE-coding genes (Fig. data) was compared between TCGA ERBB2-negative and ERBB2-positive patients’ tissue samples. Data are Mean ± SEM. *** p < 0.001 using Student’s t-test with Welch’s correction. Right panel: the interaction between the ARE-mRNA cluster or all mRNAs with ERBB2-positive gene expression data is represented by an odd ratio using Fisher’s exact test (Oncomine software). D Total RNA from the ERBB2-positive SKBR3 cell line and MCF10A normal-like ERBB2-negative cell lines were extracted and subjected to RT-QPCR using specific primers to the indicated genes. Inset shows an immunoblot of two for the ERBB2 protein among different types of breast cancer cell lines. E The SKBR3 cells were transfected with 200 nM control siRNA or ERBB2 siRNA for 24 h, followed by RNA extraction and RT-QPCR as indicated. Inset is a representative immunoblot of three, for the ERBB2 protein. The data in D and E are normalized to the housekeeping gene, RPL0. Data are Mean ± SEM (triplicate) from one representative experiment of at least two. ANOVA test demonstrates overall statistical significance. The student’s t-test was used to compare expression data for each gene.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Amplification, Functional Assay, Expressing, Software, Quantitative RT-PCR, Western Blot, Transfection, RNA Extraction

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A SKBR3 cells were treated with increasing doses of lapatinib for 24 h, and lysed for immunoblotting with anti-phospho ERBB2 protein and β-actin as a loading control. WB is one representative from three blots. B RT-QPCR expression analysis for the ERBB2-regulated ARE-mRNAs in SKBR3 cells (left panel) or MCF-10A cells ( right panel ) treated with DMSO or Lapatinib (300 nM) for overnight. Data are normalized to the housekeeping gene, RPL0. The normalized values were used to calculate % remaining relative to DMSO control. Statistical analysis was performed using two-tailed Student’s t -test for each gene reduced in expression in comparison to DMSO data. Data are Mean ± SEM (triplicate of one experiment of three). ** P < 0.001, *** P < 0.0001. C RT-QPCR for Nanoluciferase mRNA abundance or Nanoluciferase protein activity ( D ) in SKBR3 cells transfected with nLuc-ARE or control nLuc-non-ARE expression plasmid for overnight, and then treated with DMSO (control) or lapatinib for 6 h or 24 h, respectively. Data are presented as the mean ± SEM ( n = 2 experiments). ANOVA is used to compare overall significance while post hoc sidak’s test compares the DMSO and lapatinib data. *** p < 0.0001. ** p < 0.005, Student’s test. E The mRNA half-life determination in response to lapatinib. The SKBR3 cells were treated with DMSO or lapatinib (300 nM) overnight, then actinomycin D (AcD) (5 μg/ml) was added for the indicated time points. Total RNA was extracted, and cDNA samples were subjected to RT-QPCR using specific primers to the indicated genes. One-phase exponential decay curves for relative mRNA half-life measurements in the two treated groups are shown. Data are normalized to RPL0 transcript abundance, and the normalized values were used to calculate the mRNA half-life as a percentage to time 0. Data are presented as the mean ± SEM ( n = 2 experiments). F , G Immunoblot analysis for CDC6 and NEK2 protein abundance in SKBR3 cells treated as in ( A ). Representative blot each is from at least two experiments is shown.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Two Tailed Test, Activity Assay, Transfection, Plasmid Preparation

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A immunoblot analysis for ZFP36/TTP phosphorylated protein abundance in MCF10A and SKBR3 using specific primers to ZFP36/TTP and β-Actin protein as a loading control. B SKBR3 cells were transfected with an empty vector or ZFP36/TTP expression vector. Left panel: proteins were extracted, and immunoblotting was performed using a ZFP36/TTP antibody. Right panel: protein lysates were first treated with the phosphatase, CIP, or CIP phosStop C before immunoblotting with anti-ZFP36/TTP. The WB shows both phosphorylated and unphosphorylated bands. WB was performed with low exposure to avoid high background and overexposed signal of the transfected TTP (lower panel). D) SKBR3 cells were transfected with 200 nM ERBB2 siRNA or control siRNA for overnight, and ERBB2 and ZFP36/TTP protein abundance were assessed by immunoblotting. A representative blot from at least two experiments is shown. Immunoblot analysis of ERBB2 and ZFP36/TTP protein phosphorylation abundance in MCF10A ( E ) or MCF-7 ( F ) transfected with ERBB2 expression vector or empty vector. Representative blots are from at least three experiments.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A Dose-dependent activity of lapatinib on the abundance of phosphorylated ZFP36/TTP in the ERBB2-positive SKBR3 cells. The relative amounts of the proteins from three immunoblots were quantified using Image J software ( left panel ). Fold changes (Mean ± SEM, n = 3) of phosphorylated ZFP36/TTP abundance normalized to β-Actin using Image J program (right panel). B Effect of ERBB2 inhibition on ZFP36/TTP protein stability. SKBR3 cells were treated with lapatinib (300 nM) for 16 h, followed by 10 µg/ml of cycloheximide (CHX) treatment for the indicated durations (left panel). Protein lysates were subjected to immunoblotting with anti-TTP. Right panel: quantification of ZFP36/TTP abundance, normalized to GAPDH, was determined using ImageJ software and the protein half-life was assessed using the Graph Prism. Data are mean +/− SEM of two experiments. C Effect of lapatinib on the abundance of ZFP36/TTP protein in the ERBB2-positive cells, BT-474. WB is a representative of two blots from independent experiments.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Activity Assay, Western Blot, Software, Inhibition

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A Immunoblot analysis of ERBB2, AKT, MEK1/2, and ERK1/2 signaling inhibition after treatment with increasing doses of lapatinib for 24 h. Status of ERBB2 (phospho-tyrosine 1221/1222 and total), AKT (phospho-serine 473 and total pan AKT), ERK1/2 (Phospho-threonine 202/phospho-tyrosine 204 and total), MEK1/2 (Ser217/221), and ZFP36/TTP are shown. A blot from at least two experiments is shown. B Immunoblot analysis for the inhibition of MK2 activation after treatment with lapatinib. SKBR3 cells were treated with increasing doses of lapatinib, as indicated for 24 h. Phospho-threonine MK2 (T222) and total MK2 protein abundance are shown. A representative blot from two experiments is shown. C Immunoblot analysis for ZFP36/TTP phosphorylation in MAPKAPK-2 silenced SKBR3 cells. SKBR3 cells transfected with 100 nM of si Ctrl or si MAPKAPK-2 for 24 h were tested for ZFP36/TTP protein abundance. Total MK2, phosphorylated ZFP36/TTP, and β-actin protein abundances are shown. A representative blot from three experiments is shown. Effect of the MK2 inhibitor PF-3644022 (1 µM, 4 h) on the abundance of phosphorylated ZFP36/TTP in ERBB2-expressing SKBR3 ( D ) or SKBR3 transfected with ZFP36 expression plasmid ( E ). Western blots are from two independent experiments. F Immunoblot analysis of total and phosphorylated ZFP36/TTP, and MK2 in MK2 -knockout MEF cells in the absence or presence of overnight transfected ERBB2.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Western Blot, Inhibition, Activation Assay, Transfection, Expressing, Plasmid Preparation, Knock-Out

Journal: Oncogenesis

Article Title: Post-transcriptional screen of cancer amplified genes identifies ERBB2 / Her2 signaling as AU-rich mRNA stability-promoting pathway

doi: 10.1038/s41389-021-00351-w

Figure Lengend Snippet: A HEK293 cells were co-transfected with ERBB2 -HA along with ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. Proteins were extracted, and immunoblotting was performed using antibodies to ERBB2, ZFP36/TTP, and β-actin. A representative blot from three experiments is shown. B Total RNA samples were extracted and subjected to RT-QPCR using specific primers to the ERBB2-regulated ARE-mRNA s (as in Fig. D, E). Data are Mean ± SEM of average 18SRNA normalized expression of ARE-mRNA abundance remaining from vector control. C , D RT-QPCR using specific primers to the indicated genes RT-QPCR using specific primers to the indicated genes. Data in B to D are Mean ± SEM from an experiment with triplicate reactions of two independent experiments. Two-way ANOVA with Tukey’s multiple tests between the control and treatment test as indicated were used. *** p < 0.001. E Immunoblot analysis for ZFP36/TTP protein in HEK293 cells over-expressing cancer-amplified genes. HEK293 cells were co-transfected with cancer amplified genes and ZFP36/TTP -HA or vector control (0.5 µg each) for 24 h. The results of the two experiments are shown.

Article Snippet: The indicated plasmids of the amplified genes, including ERBB2-HA, were purchased from Sino Biologicals (China).

Techniques: Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Expressing, Amplification

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