p erbb2 tyr1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p erbb2 tyr1248
    Primer sequences used for the qPCR analysis
    P Erbb2 Tyr1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin"

    Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

    Journal: Drug Design, Development and Therapy

    doi: 10.2147/DDDT.S151511

    Primer sequences used for the qPCR analysis
    Figure Legend Snippet: Primer sequences used for the qPCR analysis

    Techniques Used: Amplification

    Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.
    Figure Legend Snippet: Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

    Techniques Used: Expressing

    p her2 erbb2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2 antibody
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and <t>Y1248b,</t> Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    P Her2 Erbb2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction"

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067813

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
    Figure Legend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Techniques Used: Western Blot

    p her2 tyr1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 tyr1248
    P Her2 Tyr1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p egfr  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p egfr
    Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of <t>EGFR</t> <t>and</t> <t>p-EGFR</t> was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).
    P Egfr, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Inhibition of MEIS3 Generates Cetuximab Resistance through c-Met and Akt"

    Article Title: Inhibition of MEIS3 Generates Cetuximab Resistance through c-Met and Akt

    Journal: BioMed Research International

    doi: 10.1155/2020/2046248

    Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of EGFR and p-EGFR was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).
    Figure Legend Snippet: Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of EGFR and p-EGFR was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).

    Techniques Used: shRNA, Expressing, Western Blot

    (a) Protein levels of LOVO-CON, MEIS3 shRNA, MEIS3 shRNA+c-Met overexpression, MEIS3 shRNA+c-Met shRNA, and MEIS3 overexpression+c-Met shRNA were extracted, and the expression of p-EGFR, EGFR, p-Akt, Akt, p-c-Met, and c-Met at the protein level was detected by western blot. (b) The LOVO and LOVO MEIS3 shRNA were treated with CHX for different times, and the expression of Akt was detected by western blot. (c) The SNU-61 cell line was treated with CHX for different times, and the expression of Akt was detected by western blot. (d) The LOVO cells were first treated with CHX followed by DMSO, BAF, and MG132 for different times, and the expression of Akt was determined by western blot. (e) SNU-61 (A) and HCT-116 (B) were transfected with CON and MEIS3 shRNA and then treated with DMSO, MG132, and BAF, and the degradation of Akt was determined by western blot. (f) LOVO (A) and SNU-61 (B) were transfected with the CON and MEIS3 vectors, and the ubiquitin-bonded Akt was detected using IP. (g) The LOVO cell line was transfected with random shRNA, CON vector, and c-Met shRNA for 48 h, and then, western blot was used to detect the change of c-Met in the protein level.
    Figure Legend Snippet: (a) Protein levels of LOVO-CON, MEIS3 shRNA, MEIS3 shRNA+c-Met overexpression, MEIS3 shRNA+c-Met shRNA, and MEIS3 overexpression+c-Met shRNA were extracted, and the expression of p-EGFR, EGFR, p-Akt, Akt, p-c-Met, and c-Met at the protein level was detected by western blot. (b) The LOVO and LOVO MEIS3 shRNA were treated with CHX for different times, and the expression of Akt was detected by western blot. (c) The SNU-61 cell line was treated with CHX for different times, and the expression of Akt was detected by western blot. (d) The LOVO cells were first treated with CHX followed by DMSO, BAF, and MG132 for different times, and the expression of Akt was determined by western blot. (e) SNU-61 (A) and HCT-116 (B) were transfected with CON and MEIS3 shRNA and then treated with DMSO, MG132, and BAF, and the degradation of Akt was determined by western blot. (f) LOVO (A) and SNU-61 (B) were transfected with the CON and MEIS3 vectors, and the ubiquitin-bonded Akt was detected using IP. (g) The LOVO cell line was transfected with random shRNA, CON vector, and c-Met shRNA for 48 h, and then, western blot was used to detect the change of c-Met in the protein level.

    Techniques Used: shRNA, Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation

    p neu  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p neu
    P Neu, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p her2 erbb2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 erbb2 antibody
    a ) Representative Western blots showing levels of phosphorylated <t>erbB2</t> at Y877, Y1248, Y1248-a (which represents detection of Y1248 using an alternative antibody (p- erbB2-Antibody <t>(Tyr1248)/EGFR</t> (Tyr1173)) and Y12221/2 as well as total erbB2 (t-erbB2) and actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG825 (+AG825). b ) quantification of erbB2 expression relative to actin and c–f ) quantification of erbB2 phosphorylation at the stated tyrosine site relative to total erbB2 expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).
    P Her2 Erbb2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Activation of EGFR/ERBB2 via Pathways Involving ERK1/2, P38 MAPK, AKT and FOXO Enhances Recovery of Diabetic Hearts from Ischemia-Reperfusion Injury"

    Article Title: Activation of EGFR/ERBB2 via Pathways Involving ERK1/2, P38 MAPK, AKT and FOXO Enhances Recovery of Diabetic Hearts from Ischemia-Reperfusion Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039066

    a ) Representative Western blots showing levels of phosphorylated erbB2 at Y877, Y1248, Y1248-a (which represents detection of Y1248 using an alternative antibody (p- erbB2-Antibody (Tyr1248)/EGFR (Tyr1173)) and Y12221/2 as well as total erbB2 (t-erbB2) and actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG825 (+AG825). b ) quantification of erbB2 expression relative to actin and c–f ) quantification of erbB2 phosphorylation at the stated tyrosine site relative to total erbB2 expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).
    Figure Legend Snippet: a ) Representative Western blots showing levels of phosphorylated erbB2 at Y877, Y1248, Y1248-a (which represents detection of Y1248 using an alternative antibody (p- erbB2-Antibody (Tyr1248)/EGFR (Tyr1173)) and Y12221/2 as well as total erbB2 (t-erbB2) and actin as a control protein in non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG825 (+AG825). b ) quantification of erbB2 expression relative to actin and c–f ) quantification of erbB2 phosphorylation at the stated tyrosine site relative to total erbB2 expression for all the groups studied by densitometry. N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).

    Techniques Used: Western Blot, Expressing

    Panel a) are represenatative Western Blots following immunoprecipitations (IP) with either total-EGFR or total-erbB2 antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG1478 (+AG1478) or AG825 (+AG825). N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).
    Figure Legend Snippet: Panel a) are represenatative Western Blots following immunoprecipitations (IP) with either total-EGFR or total-erbB2 antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic control hearts (C), diabetic hearts (D) and diabetic hearts chronically treated with AG1478 (+AG1478) or AG825 (+AG825). N=4; * significantly different from control (p<0.05); ** significantly different from diabetes (p<0.05).

    Techniques Used: Western Blot

    Representative Western blots to show ischemia-induced changes in phosphorylation of EGFR and erbB2 receptors and downstream signaling molecules for a) normal (non-diabetic) control hearts (C) and those subjected to 40 min ischemia (CI) and b) diabetic hearts (D) and those subjected to 40 min ischemia (DI).
    Figure Legend Snippet: Representative Western blots to show ischemia-induced changes in phosphorylation of EGFR and erbB2 receptors and downstream signaling molecules for a) normal (non-diabetic) control hearts (C) and those subjected to 40 min ischemia (CI) and b) diabetic hearts (D) and those subjected to 40 min ischemia (DI).

    Techniques Used: Western Blot

    Diabetes and/or hyperglycemia via attenuation of the EGFR/erbB2 signaling and through subsequent modulation of downstream effectors such as ERK1/2, p38 MAPK or AKT/FOXO can lead to cardiac dysfunction. The effects of diabetes on EGFR/erbB2 pathway are exacerbated by blockade of this pathway by AG1478 or AG825 which leads to worsening cardiac recovery from I/R. However, the inhibitory effects of diabetes on EGFR/ErbB2 pathway may be opposed by administering EGF that also leads to improved cardiac function. The Angiotensin II (Ang II)/AT 1 receptor pathway can also activate EGFR/erbB2 pathway that can be blocked by Losartan. Co-administration of EGF with Losartan attenuates losartan-mediated EGFR blockade and improves cardiac function in diabetes beyond that attained by either drug alone.
    Figure Legend Snippet: Diabetes and/or hyperglycemia via attenuation of the EGFR/erbB2 signaling and through subsequent modulation of downstream effectors such as ERK1/2, p38 MAPK or AKT/FOXO can lead to cardiac dysfunction. The effects of diabetes on EGFR/erbB2 pathway are exacerbated by blockade of this pathway by AG1478 or AG825 which leads to worsening cardiac recovery from I/R. However, the inhibitory effects of diabetes on EGFR/ErbB2 pathway may be opposed by administering EGF that also leads to improved cardiac function. The Angiotensin II (Ang II)/AT 1 receptor pathway can also activate EGFR/erbB2 pathway that can be blocked by Losartan. Co-administration of EGF with Losartan attenuates losartan-mediated EGFR blockade and improves cardiac function in diabetes beyond that attained by either drug alone.

    Techniques Used:

    p her2 t1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p her2 t1248
    (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and <t>p-EGFR/p-HER2</t> levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).
    P Her2 T1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "BET inhibition induces vulnerability to MCL1 targeting through upregulation of fatty acid synthesis pathway in breast cancer"

    Article Title: BET inhibition induces vulnerability to MCL1 targeting through upregulation of fatty acid synthesis pathway in breast cancer

    Journal: Cell reports

    doi: 10.1016/j.celrep.2022.111304

    (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and p-EGFR/p-HER2 levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).
    Figure Legend Snippet: (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and p-EGFR/p-HER2 levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).

    Techniques Used: Activity Assay, RNA Sequencing Assay, In Vivo, Expressing, ChIP-sequencing, Binding Assay, Amplification

    (A) Representative images (20×, bright field) of cellular morphology changes in response to BETi (48 h, 1 μM JQ1). (B) The BETi induces increased motility of HCC1954 (top) and no substantial difference in SKOV3 (bottom) as monitored across time points with a wound scratching assay. (C) Drug-induced changes in membrane fluidity (mean of triplicates, error bars represent ±SEM) in HCC1954 and SKOV3 cells based on fluorescent lipophilic pyrene probes that are enriched in excimer state (emission = 470 nm) with increasing membrane fluidity and enriched in monomers (emission = 400 nm) with decreasing membrane fluidity. (D) The representative images of HER2/EGFR localization changes in response to BET inhibition (48 h, 1 μM). Images are collected using fluorescent confocal microscopy (20×). The fluorescence signals from EGFR/HER2 (green) in BETi-treated HCC1954 cells suggest increased localization of the receptors. The EGFR/HER2 signal was unchanged in response to BETi in SKOV3. (E) The proposed mechanism of BET inhibition-induced vulnerability to BET and MCL1 co-targeting in the context of MCL1 chromosomal amplifications or gains based on the integrated multi-omics, network, and perturbation analyses. The marked genes and proteins are identified and validated in molecular perturbation response analyses.
    Figure Legend Snippet: (A) Representative images (20×, bright field) of cellular morphology changes in response to BETi (48 h, 1 μM JQ1). (B) The BETi induces increased motility of HCC1954 (top) and no substantial difference in SKOV3 (bottom) as monitored across time points with a wound scratching assay. (C) Drug-induced changes in membrane fluidity (mean of triplicates, error bars represent ±SEM) in HCC1954 and SKOV3 cells based on fluorescent lipophilic pyrene probes that are enriched in excimer state (emission = 470 nm) with increasing membrane fluidity and enriched in monomers (emission = 400 nm) with decreasing membrane fluidity. (D) The representative images of HER2/EGFR localization changes in response to BET inhibition (48 h, 1 μM). Images are collected using fluorescent confocal microscopy (20×). The fluorescence signals from EGFR/HER2 (green) in BETi-treated HCC1954 cells suggest increased localization of the receptors. The EGFR/HER2 signal was unchanged in response to BETi in SKOV3. (E) The proposed mechanism of BET inhibition-induced vulnerability to BET and MCL1 co-targeting in the context of MCL1 chromosomal amplifications or gains based on the integrated multi-omics, network, and perturbation analyses. The marked genes and proteins are identified and validated in molecular perturbation response analyses.

    Techniques Used: Inhibition, Confocal Microscopy, Fluorescence

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Viability Assay, Transfection, SYBR Green Assay, Plasmid Preparation, Electron Microscopy, Expressing, Negative Control, Software, Flow Cytometry

    p erbb2 tyr 1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p erbb2 tyr 1248
    P Erbb2 Tyr 1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti p her2 y1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti p her2 y1248
    (A) Schematic depiction of the mutations drawn on cartoon representations of the kinase domains of <t>HER2</t> and HER3. Amino acid (aa) numbering is based on the nascent reading frame. (B) CHO-K1 cells were cotransfected to overexpress HER2 and to express HER3, such as to generate constitutive HER2 autophosphorylation (lane 1) and HER3 transphosphorylation (lane 5). After 24 h, cell signaling was assayed as shown. Wild-type (WT) constructs were compared with mutants in the indicated combinations. The HER2 N-lobe mutant (I714Q) is a defective allosteric receiver, whereas the HER2 C-lobe mutant (V956R) is an impaired allosteric activator but competent receiver. HER3 is catalytically inactive and thus an obligate allosteric activator, and the HER3 C-lobe mutant (V945R) is an impaired allosteric activator. (C) CHO cells were transfected as in (B) using WT HER2 and HER3 in additional combinations. The HER2 C-lobe mutant (V956R) is an impaired allosteric activator. The HER3 single C-lobe mutant (V945R) is an impaired allosteric activator but retains some activation function. The HER3 triple C-lobe mutant (I938R-V945R-M949R) is an even more impaired allosteric activator. (D) CHO cells were transfected as in (B). The HER2 C-lobe activation function can similarly be further impaired by triple mutation. The double banding of HER3 observed in these CHO-K1 cell transfections is due to differential glycosylation . (E) CHO cells were cotransfected to overexpress HER2 and express HER3 and were treated with DMSO or either of three small molecules that bind the HER3 KD with high affinity. These were administered at 1 μM for 1 h. The binding K D s for HER3 binding are as follows: bosutinib 0.8 nM, PP-242 120 nM, and dasatinib 18 nM . The affinities for HER2 KD are >1,400 nM for these drugs. The dephosphorylation of Src is shown as a positive control for bosutinib and dasatinib, which also inhibit Src. (F) CHO-K1 cells were transfected as in (A) using WT or mutant HER2 and HER3 constructs. The HER3 K742M mutant is defective at ATP binding. Lane 8 was treated with 1 μM lapatinib for 1 h. This image was cropped down from 13 lanes for clarity. (G) CHO-K1 cells were transfected as in (C) using WT or mutant HER2 and HER3 constructs.
    Anti P Her2 Y1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers"

    Article Title: Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.110291

    (A) Schematic depiction of the mutations drawn on cartoon representations of the kinase domains of HER2 and HER3. Amino acid (aa) numbering is based on the nascent reading frame. (B) CHO-K1 cells were cotransfected to overexpress HER2 and to express HER3, such as to generate constitutive HER2 autophosphorylation (lane 1) and HER3 transphosphorylation (lane 5). After 24 h, cell signaling was assayed as shown. Wild-type (WT) constructs were compared with mutants in the indicated combinations. The HER2 N-lobe mutant (I714Q) is a defective allosteric receiver, whereas the HER2 C-lobe mutant (V956R) is an impaired allosteric activator but competent receiver. HER3 is catalytically inactive and thus an obligate allosteric activator, and the HER3 C-lobe mutant (V945R) is an impaired allosteric activator. (C) CHO cells were transfected as in (B) using WT HER2 and HER3 in additional combinations. The HER2 C-lobe mutant (V956R) is an impaired allosteric activator. The HER3 single C-lobe mutant (V945R) is an impaired allosteric activator but retains some activation function. The HER3 triple C-lobe mutant (I938R-V945R-M949R) is an even more impaired allosteric activator. (D) CHO cells were transfected as in (B). The HER2 C-lobe activation function can similarly be further impaired by triple mutation. The double banding of HER3 observed in these CHO-K1 cell transfections is due to differential glycosylation . (E) CHO cells were cotransfected to overexpress HER2 and express HER3 and were treated with DMSO or either of three small molecules that bind the HER3 KD with high affinity. These were administered at 1 μM for 1 h. The binding K D s for HER3 binding are as follows: bosutinib 0.8 nM, PP-242 120 nM, and dasatinib 18 nM . The affinities for HER2 KD are >1,400 nM for these drugs. The dephosphorylation of Src is shown as a positive control for bosutinib and dasatinib, which also inhibit Src. (F) CHO-K1 cells were transfected as in (A) using WT or mutant HER2 and HER3 constructs. The HER3 K742M mutant is defective at ATP binding. Lane 8 was treated with 1 μM lapatinib for 1 h. This image was cropped down from 13 lanes for clarity. (G) CHO-K1 cells were transfected as in (C) using WT or mutant HER2 and HER3 constructs.
    Figure Legend Snippet: (A) Schematic depiction of the mutations drawn on cartoon representations of the kinase domains of HER2 and HER3. Amino acid (aa) numbering is based on the nascent reading frame. (B) CHO-K1 cells were cotransfected to overexpress HER2 and to express HER3, such as to generate constitutive HER2 autophosphorylation (lane 1) and HER3 transphosphorylation (lane 5). After 24 h, cell signaling was assayed as shown. Wild-type (WT) constructs were compared with mutants in the indicated combinations. The HER2 N-lobe mutant (I714Q) is a defective allosteric receiver, whereas the HER2 C-lobe mutant (V956R) is an impaired allosteric activator but competent receiver. HER3 is catalytically inactive and thus an obligate allosteric activator, and the HER3 C-lobe mutant (V945R) is an impaired allosteric activator. (C) CHO cells were transfected as in (B) using WT HER2 and HER3 in additional combinations. The HER2 C-lobe mutant (V956R) is an impaired allosteric activator. The HER3 single C-lobe mutant (V945R) is an impaired allosteric activator but retains some activation function. The HER3 triple C-lobe mutant (I938R-V945R-M949R) is an even more impaired allosteric activator. (D) CHO cells were transfected as in (B). The HER2 C-lobe activation function can similarly be further impaired by triple mutation. The double banding of HER3 observed in these CHO-K1 cell transfections is due to differential glycosylation . (E) CHO cells were cotransfected to overexpress HER2 and express HER3 and were treated with DMSO or either of three small molecules that bind the HER3 KD with high affinity. These were administered at 1 μM for 1 h. The binding K D s for HER3 binding are as follows: bosutinib 0.8 nM, PP-242 120 nM, and dasatinib 18 nM . The affinities for HER2 KD are >1,400 nM for these drugs. The dephosphorylation of Src is shown as a positive control for bosutinib and dasatinib, which also inhibit Src. (F) CHO-K1 cells were transfected as in (A) using WT or mutant HER2 and HER3 constructs. The HER3 K742M mutant is defective at ATP binding. Lane 8 was treated with 1 μM lapatinib for 1 h. This image was cropped down from 13 lanes for clarity. (G) CHO-K1 cells were transfected as in (C) using WT or mutant HER2 and HER3 constructs.

    Techniques Used: Construct, Mutagenesis, Transfection, Activation Assay, Binding Assay, De-Phosphorylation Assay, Positive Control

    (A) RMSD plot from MD simulations of the ATP-bound and apo HER3 pseudokinase states. (B) The superimposed cartoon images of the ATP-bound and apo HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (C) RMSD plot from MD simulations of the ATP-bound WT, apo WT, and apo K742M HER3 pseudokinase states. (D) The superimposed cartoon images of the ATP-bound WT, apo WT, and apo K742M HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (E) Left: plot of mutual information in kcal/mol between the transmitter (AP-2 pocket: residues 683, 685, 689, 690, 709, 718, 720, 756, and 767; side chains shown in green in right panel) and receiver (P loop: residues 696–704; side chains shown in yellow in right panel) sites, showing ~2 kcal/mol mutual information in both the absence (WT apo) and presence (WT ATP) of ATP. Error bars reflect standard deviation. Right: illustration of the normalized per-residue contribution to co-information in apo and ATP-bound simulations of HER3; “min” and “max” refer to the minimum and maximum values among all possible residues across all simulations. Residues colored dark red contribute most to communication between the AP-2 pocket and the P loop. (F) CHO-K1 cells were transfected to overexpress HER2 and to express the indicated WT or mutant HER3 constructs. The surface residues F704, L709, and V786 were mutated to impair the binding affinity of the AP-2 pocket. (G) W728 was mutated to collapse the AP-2 pocket. (H) The effect of ligand stimulation was studied on the F704 AP-2 pocket mutant.
    Figure Legend Snippet: (A) RMSD plot from MD simulations of the ATP-bound and apo HER3 pseudokinase states. (B) The superimposed cartoon images of the ATP-bound and apo HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (C) RMSD plot from MD simulations of the ATP-bound WT, apo WT, and apo K742M HER3 pseudokinase states. (D) The superimposed cartoon images of the ATP-bound WT, apo WT, and apo K742M HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (E) Left: plot of mutual information in kcal/mol between the transmitter (AP-2 pocket: residues 683, 685, 689, 690, 709, 718, 720, 756, and 767; side chains shown in green in right panel) and receiver (P loop: residues 696–704; side chains shown in yellow in right panel) sites, showing ~2 kcal/mol mutual information in both the absence (WT apo) and presence (WT ATP) of ATP. Error bars reflect standard deviation. Right: illustration of the normalized per-residue contribution to co-information in apo and ATP-bound simulations of HER3; “min” and “max” refer to the minimum and maximum values among all possible residues across all simulations. Residues colored dark red contribute most to communication between the AP-2 pocket and the P loop. (F) CHO-K1 cells were transfected to overexpress HER2 and to express the indicated WT or mutant HER3 constructs. The surface residues F704, L709, and V786 were mutated to impair the binding affinity of the AP-2 pocket. (G) W728 was mutated to collapse the AP-2 pocket. (H) The effect of ligand stimulation was studied on the F704 AP-2 pocket mutant.

    Techniques Used: Standard Deviation, Transfection, Mutagenesis, Construct, Binding Assay

    (A) HCC1569 human HER2-amplified breast cancer cells were engineered to eliminate HER3 expression by CRISPR-Cas targeting (HCC1569-HER3KO). To eliminate the role of clonal growth characteristics in the replacement experiments, we mixed together three separate clones of HCC1569-HER3KO cells to generate a polyclonal HCC1569-HER3KO cell line (lane 2), and this cell line was used as the parental cell line for the various add-back experiments. These were then transduced to re-express WT HER3 (lane 4) or experimental mutant HER3 constructs. Experimental add-backs included a HER3 C-lobe mutant defective at allosteric activation (HER3 I938R/V945R/M949R) and a HER3 with a mutated AP-2 pocket (F704D). The expression of firefly luciferase (lane 3) constitutes a negative control cell type. The add-back HER3 constructs contain C-terminal myc tags. (B) The indicated engineered versions of HER2-amplified HCC1569 tumor cells were inoculated subcutaneously in NSG mice, and tumor volumes were measured over time. The number of surviving mice along the time course of the animal studies is shown for each arm underneath, and the sample size reduction over time in some arms reflects the removal of mice for euthanasia because of large tumor sizes as mandated by guidelines. The error bars reflect SEM.
    Figure Legend Snippet: (A) HCC1569 human HER2-amplified breast cancer cells were engineered to eliminate HER3 expression by CRISPR-Cas targeting (HCC1569-HER3KO). To eliminate the role of clonal growth characteristics in the replacement experiments, we mixed together three separate clones of HCC1569-HER3KO cells to generate a polyclonal HCC1569-HER3KO cell line (lane 2), and this cell line was used as the parental cell line for the various add-back experiments. These were then transduced to re-express WT HER3 (lane 4) or experimental mutant HER3 constructs. Experimental add-backs included a HER3 C-lobe mutant defective at allosteric activation (HER3 I938R/V945R/M949R) and a HER3 with a mutated AP-2 pocket (F704D). The expression of firefly luciferase (lane 3) constitutes a negative control cell type. The add-back HER3 constructs contain C-terminal myc tags. (B) The indicated engineered versions of HER2-amplified HCC1569 tumor cells were inoculated subcutaneously in NSG mice, and tumor volumes were measured over time. The number of surviving mice along the time course of the animal studies is shown for each arm underneath, and the sample size reduction over time in some arms reflects the removal of mice for euthanasia because of large tumor sizes as mandated by guidelines. The error bars reflect SEM.

    Techniques Used: Amplification, Expressing, CRISPR, Clone Assay, Mutagenesis, Construct, Activation Assay, Luciferase, Negative Control

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Transfection, Plasmid Preparation, BIA-KA, Mutagenesis, Modification, Software, Expressing

    anti p her2 y1248  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti p her2 y1248
    (A) The ECD of HER3 was locked in the closed/tethered conformation by introducing double cysteine mutations at the indicated nearby residues of domains II and IV. Three different locked versions of HER3 were generated by mutating the indicated pairs of residues to cysteines as indicated. Of these, the Y265C/V593C double mutant has been studied extensively before and confirmed to be locked in the closed conformation by disulfide bridging . (B)CHO-K1 cells were transfected to express <t>HER2</t> and HER3 mutants as indicated. Wild-type HER3 is constitutively phosphorylated in the presence of overexpressed HER2 (lane 1)and is further inducible by ligand stimulation (lane 2). There is also slight induction of HER3 phosphorylation by the background low level of endogenous HER2 in CHO-K1 cells (lane 4). The locked HER3 mutants, in the presence of overexpressed HER2, are fully competent at constitutive phosphorylation despite the fact that they are not able to adopt the open conformation and expose their dimerization interface and have lost ligand responsiveness (lanes 5–16). (C) The entire ECDs of HER2 and HER3 were deleted, preserving the N-terminal signal sequences. Membrane localization of these constructs was confirmed by immunofluorescence staining as indicated. (D) These constructs are fully competent at constitutive phosphorylation when HER2 is overexpressed, despite complete loss of ECD functions.
    Anti P Her2 Y1248, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Extensive conformational and physical plasticity protects HER2-HER3 tumorigenic signaling"

    Article Title: Extensive conformational and physical plasticity protects HER2-HER3 tumorigenic signaling

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.110285

    (A) The ECD of HER3 was locked in the closed/tethered conformation by introducing double cysteine mutations at the indicated nearby residues of domains II and IV. Three different locked versions of HER3 were generated by mutating the indicated pairs of residues to cysteines as indicated. Of these, the Y265C/V593C double mutant has been studied extensively before and confirmed to be locked in the closed conformation by disulfide bridging . (B)CHO-K1 cells were transfected to express HER2 and HER3 mutants as indicated. Wild-type HER3 is constitutively phosphorylated in the presence of overexpressed HER2 (lane 1)and is further inducible by ligand stimulation (lane 2). There is also slight induction of HER3 phosphorylation by the background low level of endogenous HER2 in CHO-K1 cells (lane 4). The locked HER3 mutants, in the presence of overexpressed HER2, are fully competent at constitutive phosphorylation despite the fact that they are not able to adopt the open conformation and expose their dimerization interface and have lost ligand responsiveness (lanes 5–16). (C) The entire ECDs of HER2 and HER3 were deleted, preserving the N-terminal signal sequences. Membrane localization of these constructs was confirmed by immunofluorescence staining as indicated. (D) These constructs are fully competent at constitutive phosphorylation when HER2 is overexpressed, despite complete loss of ECD functions.
    Figure Legend Snippet: (A) The ECD of HER3 was locked in the closed/tethered conformation by introducing double cysteine mutations at the indicated nearby residues of domains II and IV. Three different locked versions of HER3 were generated by mutating the indicated pairs of residues to cysteines as indicated. Of these, the Y265C/V593C double mutant has been studied extensively before and confirmed to be locked in the closed conformation by disulfide bridging . (B)CHO-K1 cells were transfected to express HER2 and HER3 mutants as indicated. Wild-type HER3 is constitutively phosphorylated in the presence of overexpressed HER2 (lane 1)and is further inducible by ligand stimulation (lane 2). There is also slight induction of HER3 phosphorylation by the background low level of endogenous HER2 in CHO-K1 cells (lane 4). The locked HER3 mutants, in the presence of overexpressed HER2, are fully competent at constitutive phosphorylation despite the fact that they are not able to adopt the open conformation and expose their dimerization interface and have lost ligand responsiveness (lanes 5–16). (C) The entire ECDs of HER2 and HER3 were deleted, preserving the N-terminal signal sequences. Membrane localization of these constructs was confirmed by immunofluorescence staining as indicated. (D) These constructs are fully competent at constitutive phosphorylation when HER2 is overexpressed, despite complete loss of ECD functions.

    Techniques Used: Generated, Mutagenesis, Transfection, Preserving, Construct, Immunofluorescence, Staining

    (A) High affinity IgG binding residues from the Ig-binding domain of streptococcal protein G were inserted into the indicated G1 or G2 linker regions of the HER3 ECD. (B) The HER3-G1 and HER3-G2 mutants were expressed in CHO-K1 cells along with overexpressed HER2 and assayed as shown. The altered HER3 constructs are myc tagged. When cultured in medium, the abundant IgGs in bovine serum bind the engineered HER3 constructs. The G1* and G2* constructs are negative control versions of the G1 and G2 constructs, mutated within the protein G sequence to abolish IgG binding. Despite confirmation of IgG binding, these proximity-restricting HER3 mutants are fully capable of constitutive HER2-HER3 phosphorylation in CHO-K1 cells when HER2 is overexpressed. The double banding of HER3 is due to glycosylation effects . (C) The experiment was repeated using pertuzumab, which binds HER2 at its dimerization interface.
    Figure Legend Snippet: (A) High affinity IgG binding residues from the Ig-binding domain of streptococcal protein G were inserted into the indicated G1 or G2 linker regions of the HER3 ECD. (B) The HER3-G1 and HER3-G2 mutants were expressed in CHO-K1 cells along with overexpressed HER2 and assayed as shown. The altered HER3 constructs are myc tagged. When cultured in medium, the abundant IgGs in bovine serum bind the engineered HER3 constructs. The G1* and G2* constructs are negative control versions of the G1 and G2 constructs, mutated within the protein G sequence to abolish IgG binding. Despite confirmation of IgG binding, these proximity-restricting HER3 mutants are fully capable of constitutive HER2-HER3 phosphorylation in CHO-K1 cells when HER2 is overexpressed. The double banding of HER3 is due to glycosylation effects . (C) The experiment was repeated using pertuzumab, which binds HER2 at its dimerization interface.

    Techniques Used: Binding Assay, Construct, Cell Culture, Negative Control, Sequencing

    (A) Domain 4 of the HER3 ECD was replaced with domain 4 of the HER2 ECD, creating the domain-swapped (DS) mutant of HER3. These domains are structurally homologous, but the transposition brings the trastuzumab-binding epitope to the juxtamembrane region of HER3. Trastuzumab binds its target in a conformation where it protrudes directly into the dimerization plane (PDB: 1N8Z; ), creating considerable steric clash within the dimerization plane between its two targets that would be maximally restrictive to receptor proximation. (B) Despite confirmed trastuzumab binding to HER2 and the HER3-DS mutant, constitutive HER2-HER3 signaling persists in this engineered scenario in CHO-K1 cells.
    Figure Legend Snippet: (A) Domain 4 of the HER3 ECD was replaced with domain 4 of the HER2 ECD, creating the domain-swapped (DS) mutant of HER3. These domains are structurally homologous, but the transposition brings the trastuzumab-binding epitope to the juxtamembrane region of HER3. Trastuzumab binds its target in a conformation where it protrudes directly into the dimerization plane (PDB: 1N8Z; ), creating considerable steric clash within the dimerization plane between its two targets that would be maximally restrictive to receptor proximation. (B) Despite confirmed trastuzumab binding to HER2 and the HER3-DS mutant, constitutive HER2-HER3 signaling persists in this engineered scenario in CHO-K1 cells.

    Techniques Used: Mutagenesis, Binding Assay

    (A) The ECD of HER3 was replaced with the ECD of FAIM3, the receptor for pentameric IgM, and expressed along with overexpressed HER2 in CHO-K1 cells. Despite treatment with biotinylated pentameric IgM and confirmed IgM binding to HER3, constitutive HER2-HER3 signaling persists. SkBr3 lysates were used in lane 10. (B) The ECDs of HER2 and HER3 were replaced with the ECD of FAIM3, and these hybrid receptors were expressed in CHO-K1 cells and treated with biotinylated pentameric IgM. Despite treatment with biotinylated pentameric IgM, constitutive signaling persists. The HER2* and HER3* versions in lanes 5 and 6 are negative control mutants that have additional mutations in the intracellular region that disrupt kinase domain dimerization and activation. These include mutations within the juxtamembrane regions of HER2 and HER3 and mutations in the N-lobe of the HER2 kinase domain. In lane 9, the lysates from lane 4 were used, but the pull-down control was with mouse IgG-agarose beads. The HER2 constructs are hemagglutinin (HA) tagged, and HER3 constructs are FLAG tagged.
    Figure Legend Snippet: (A) The ECD of HER3 was replaced with the ECD of FAIM3, the receptor for pentameric IgM, and expressed along with overexpressed HER2 in CHO-K1 cells. Despite treatment with biotinylated pentameric IgM and confirmed IgM binding to HER3, constitutive HER2-HER3 signaling persists. SkBr3 lysates were used in lane 10. (B) The ECDs of HER2 and HER3 were replaced with the ECD of FAIM3, and these hybrid receptors were expressed in CHO-K1 cells and treated with biotinylated pentameric IgM. Despite treatment with biotinylated pentameric IgM, constitutive signaling persists. The HER2* and HER3* versions in lanes 5 and 6 are negative control mutants that have additional mutations in the intracellular region that disrupt kinase domain dimerization and activation. These include mutations within the juxtamembrane regions of HER2 and HER3 and mutations in the N-lobe of the HER2 kinase domain. In lane 9, the lysates from lane 4 were used, but the pull-down control was with mouse IgG-agarose beads. The HER2 constructs are hemagglutinin (HA) tagged, and HER3 constructs are FLAG tagged.

    Techniques Used: Binding Assay, Negative Control, Activation Assay, Construct

    (A) The surface of CHO cells expressing HER2 and HER3 was biotinylated using a cell-impermeable reagent. The entire surface proteome was then depleted from cell lysates using streptavidin beads, and HER2-HER3 expression and signaling activity was assayed in the intracellular lysate as shown. Lane 3 shows the membrane-depleted intracellular lysate; all other lanes are various negative controls. S indicates depletion by streptavidin beads, D indicates dummy beads, and – indicates no beads. (B) The same experiment was performed on HER2-amplified HCC1569 breast cancer cells. HER3 signaling in the intracellular lysate was assayed as shown. The streptavidin immunoblot shows the total depletion of the surface proteome in experimental lane 3.
    Figure Legend Snippet: (A) The surface of CHO cells expressing HER2 and HER3 was biotinylated using a cell-impermeable reagent. The entire surface proteome was then depleted from cell lysates using streptavidin beads, and HER2-HER3 expression and signaling activity was assayed in the intracellular lysate as shown. Lane 3 shows the membrane-depleted intracellular lysate; all other lanes are various negative controls. S indicates depletion by streptavidin beads, D indicates dummy beads, and – indicates no beads. (B) The same experiment was performed on HER2-amplified HCC1569 breast cancer cells. HER3 signaling in the intracellular lysate was assayed as shown. The streptavidin immunoblot shows the total depletion of the surface proteome in experimental lane 3.

    Techniques Used: Expressing, Activity Assay, Amplification, Western Blot

    (A) HER3 was knocked out in HCC1569 cells (HCC1569HER3KO) and replaced by the indicated add-back versions. These include wild-type HER3 (positive control), firefly luciferase (negative control), and a HER3 construct with the ECD locked in the inactive conformation (Y265C/V593C; ECDlocked), or with the ECD entirely deleted and replaced by Gaussia luciferase (ECDΔgLuc). These tumor cells were implanted subcutaneously, and tumor growth was monitored in NSG mice. Although the volumes of the HCC1569HER3KO-HER3-ECD-locked tumors appear lower than those of the HER3 wild type, this is due to altered biology. This tumor grows in a more flat and diffuse pattern with frequent ulceration and loss of mice. (B) HCC1569HER3KO cells were engineered to re-express a HER3 construct with high-affinity IgG-binding residues inserted between ECD domains I-II (HER3-G1) . These tumors were grown subcutaneously in NSG mice and treated as indicated. IgG treatment targets HER3 at the engineered target site, and pertuzumab targets HER2 at its dimerization interface, but tumorigenic growth persists. (C) HCC1569HER3KO cells were engineered to re-express the DS (HER3DS) version of HER3 , which brings the trastuzumab-binding epitope to the extracellular juxtamembrane region of HER3. Trastuzumab binds the extracellular juxtamembrane regions of HER2 and HER3 in these cells, but tumor growth is unaffected. The Fc-mutated version of trastuzumab (si4D5) is used here to minimize its immunologically mediated anti-tumor effects and focus on its function-disrupting effects. (D) HCC1569HER3KO cells were engineered to express the FAIM3-HER3 hybrid receptor and grown subcutaneously in NSG mice. Treatment with pentameric IgM and trastuzumab/si4D5 does not inhibit the growth of these tumors despite the significant spherical constraints on proximation of IgM-bound HER3 with HER2. (E and F) HCC1569 tumors with wild-type HER3 (E) or the ligand nonresponsive HER3 ECDΔgLuc hybrid mutant (F) were used here to inoculate the mammary fat pads of NSG mice. When tumors reached approximately 300 mm 3 , mice were randomized to receive treatment with lapatinib (80 mg/kg/day) in two divided doses by oral gavage or vehicle control. This dose of lapatinib is below the maximal tolerated dose of 100 mg/kg/day, which effectively suppresses HCC1569 tumor growth. A submaximal dose was used to best demonstrate the supersensitivity of the ligand-nonresponsive mutant if such an effect were to be seen. The ligand-nonresponsive mutant of HER3 shows no sensitization to lapatinib compared with the wild type. The numbers of surviving mice along the time course of the animal studies are shown for each arm underneath, and the sample size reduction over time in some arms reflects removal of mice for euthanasia because of large tumors, as mandated by guidelines. The error bars reflect SEM.
    Figure Legend Snippet: (A) HER3 was knocked out in HCC1569 cells (HCC1569HER3KO) and replaced by the indicated add-back versions. These include wild-type HER3 (positive control), firefly luciferase (negative control), and a HER3 construct with the ECD locked in the inactive conformation (Y265C/V593C; ECDlocked), or with the ECD entirely deleted and replaced by Gaussia luciferase (ECDΔgLuc). These tumor cells were implanted subcutaneously, and tumor growth was monitored in NSG mice. Although the volumes of the HCC1569HER3KO-HER3-ECD-locked tumors appear lower than those of the HER3 wild type, this is due to altered biology. This tumor grows in a more flat and diffuse pattern with frequent ulceration and loss of mice. (B) HCC1569HER3KO cells were engineered to re-express a HER3 construct with high-affinity IgG-binding residues inserted between ECD domains I-II (HER3-G1) . These tumors were grown subcutaneously in NSG mice and treated as indicated. IgG treatment targets HER3 at the engineered target site, and pertuzumab targets HER2 at its dimerization interface, but tumorigenic growth persists. (C) HCC1569HER3KO cells were engineered to re-express the DS (HER3DS) version of HER3 , which brings the trastuzumab-binding epitope to the extracellular juxtamembrane region of HER3. Trastuzumab binds the extracellular juxtamembrane regions of HER2 and HER3 in these cells, but tumor growth is unaffected. The Fc-mutated version of trastuzumab (si4D5) is used here to minimize its immunologically mediated anti-tumor effects and focus on its function-disrupting effects. (D) HCC1569HER3KO cells were engineered to express the FAIM3-HER3 hybrid receptor and grown subcutaneously in NSG mice. Treatment with pentameric IgM and trastuzumab/si4D5 does not inhibit the growth of these tumors despite the significant spherical constraints on proximation of IgM-bound HER3 with HER2. (E and F) HCC1569 tumors with wild-type HER3 (E) or the ligand nonresponsive HER3 ECDΔgLuc hybrid mutant (F) were used here to inoculate the mammary fat pads of NSG mice. When tumors reached approximately 300 mm 3 , mice were randomized to receive treatment with lapatinib (80 mg/kg/day) in two divided doses by oral gavage or vehicle control. This dose of lapatinib is below the maximal tolerated dose of 100 mg/kg/day, which effectively suppresses HCC1569 tumor growth. A submaximal dose was used to best demonstrate the supersensitivity of the ligand-nonresponsive mutant if such an effect were to be seen. The ligand-nonresponsive mutant of HER3 shows no sensitization to lapatinib compared with the wild type. The numbers of surviving mice along the time course of the animal studies are shown for each arm underneath, and the sample size reduction over time in some arms reflects removal of mice for euthanasia because of large tumors, as mandated by guidelines. The error bars reflect SEM.

    Techniques Used: Positive Control, Luciferase, Negative Control, Construct, Binding Assay, Mutagenesis

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Transfection, Plasmid Preparation, BIA-KA, Mutagenesis, Modification

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    Cell Signaling Technology Inc p erbb2 tyr1248
    Primer sequences used for the qPCR analysis
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    Cell Signaling Technology Inc p her2 erbb2 antibody
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and <t>Y1248b,</t> Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
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    Cell Signaling Technology Inc p her2 tyr1248
    Panel a) is a representative Western blot showing the levels of phosphorylated <t>ErbB2</t> (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and <t>Y1248b,</t> Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).
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    Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of <t>EGFR</t> <t>and</t> <t>p-EGFR</t> was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).
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    Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of <t>EGFR</t> <t>and</t> <t>p-EGFR</t> was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).
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    Cell Signaling Technology Inc p her2 t1248
    (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and <t>p-EGFR/p-HER2</t> levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).
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    Cell Signaling Technology Inc p erbb2 tyr 1248
    (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and <t>p-EGFR/p-HER2</t> levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).
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    Cell Signaling Technology Inc anti p her2 y1248
    (A) Schematic depiction of the mutations drawn on cartoon representations of the kinase domains of <t>HER2</t> and HER3. Amino acid (aa) numbering is based on the nascent reading frame. (B) CHO-K1 cells were cotransfected to overexpress HER2 and to express HER3, such as to generate constitutive HER2 autophosphorylation (lane 1) and HER3 transphosphorylation (lane 5). After 24 h, cell signaling was assayed as shown. Wild-type (WT) constructs were compared with mutants in the indicated combinations. The HER2 N-lobe mutant (I714Q) is a defective allosteric receiver, whereas the HER2 C-lobe mutant (V956R) is an impaired allosteric activator but competent receiver. HER3 is catalytically inactive and thus an obligate allosteric activator, and the HER3 C-lobe mutant (V945R) is an impaired allosteric activator. (C) CHO cells were transfected as in (B) using WT HER2 and HER3 in additional combinations. The HER2 C-lobe mutant (V956R) is an impaired allosteric activator. The HER3 single C-lobe mutant (V945R) is an impaired allosteric activator but retains some activation function. The HER3 triple C-lobe mutant (I938R-V945R-M949R) is an even more impaired allosteric activator. (D) CHO cells were transfected as in (B). The HER2 C-lobe activation function can similarly be further impaired by triple mutation. The double banding of HER3 observed in these CHO-K1 cell transfections is due to differential glycosylation . (E) CHO cells were cotransfected to overexpress HER2 and express HER3 and were treated with DMSO or either of three small molecules that bind the HER3 KD with high affinity. These were administered at 1 μM for 1 h. The binding K D s for HER3 binding are as follows: bosutinib 0.8 nM, PP-242 120 nM, and dasatinib 18 nM . The affinities for HER2 KD are >1,400 nM for these drugs. The dephosphorylation of Src is shown as a positive control for bosutinib and dasatinib, which also inhibit Src. (F) CHO-K1 cells were transfected as in (A) using WT or mutant HER2 and HER3 constructs. The HER3 K742M mutant is defective at ATP binding. Lane 8 was treated with 1 μM lapatinib for 1 h. This image was cropped down from 13 lanes for clarity. (G) CHO-K1 cells were transfected as in (C) using WT or mutant HER2 and HER3 constructs.
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    Image Search Results


    Primer sequences used for the qPCR analysis

    Journal: Drug Design, Development and Therapy

    Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

    doi: 10.2147/DDDT.S151511

    Figure Lengend Snippet: Primer sequences used for the qPCR analysis

    Article Snippet: The following antibodies and concentrations were used over night at 4°C; NRG1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-28916; 1:400), ErbB4 (Santa Cruz Biotechnology Inc., sc-283; 1:500), ErbB2 (Cell Signaling, Danvers, MA, USA, 2165; 1:1,500), p-ErbB4 (Tyr1056) (Santa Cruz Biotechnology Inc., sc33040; 1:300), p-ErbB2 (Tyr1248) (Cell Signaling, 2247; 1:1,500), p-Akt (Ser473) (Cell Signaling, 4060; 1:3,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 4695; 1:2,000), caspase-3 (Abcam, Cambridge, UK, ab4051; 1:500), and β-actin (Proteintech, Rosemont, IL, USA, 66009-1-Ig; 1:4,000).

    Techniques: Amplification

    Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

    Journal: Drug Design, Development and Therapy

    Article Title: Dysregulation of Neuregulin-1/ErbB signaling in the hippocampus of rats after administration of doxorubicin

    doi: 10.2147/DDDT.S151511

    Figure Lengend Snippet: Effect of Dox on gene expression of ErbB2, ErbB4, and the ratio of pErbB4/ErbB4 and pErbB2/ErbB2 in the hippocampus. Notes: ErbB4 mRNA expression ( A ); pErbB4/ErbB4 ratio ( B ); ErbB2 mRNA expression ( C ); and pErbB2/ErbB2 ratio ( D ). Data are expressed as mean ± SEM (n=6–7). * p <0.05 and ** p <0.01 compared to the control group. Abbreviations: Dox, doxorubicin; DoxS, doxorubicin administration for short time; DoxL, doxorubicin administration for long time; SEM, standard error of the mean.

    Article Snippet: The following antibodies and concentrations were used over night at 4°C; NRG1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, sc-28916; 1:400), ErbB4 (Santa Cruz Biotechnology Inc., sc-283; 1:500), ErbB2 (Cell Signaling, Danvers, MA, USA, 2165; 1:1,500), p-ErbB4 (Tyr1056) (Santa Cruz Biotechnology Inc., sc33040; 1:300), p-ErbB2 (Tyr1248) (Cell Signaling, 2247; 1:1,500), p-Akt (Ser473) (Cell Signaling, 4060; 1:3,000), p-ERK (Thr202/Tyr204) (Cell Signaling, 4695; 1:2,000), caspase-3 (Abcam, Cambridge, UK, ab4051; 1:500), and β-actin (Proteintech, Rosemont, IL, USA, 66009-1-Ig; 1:4,000).

    Techniques: Expressing

    Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: Panel a) is a representative Western blot showing the levels of phosphorylated ErbB2 (p-ErbB2) at the indicated tyrosines Y1221/1222, Y1248 (detected by separate antibodies labeled as Y1248 a and Y1248b, Y877, total (t-) ErbB2 and β-actin in the isolated mesenteric bed from normal controls (C), diabetic (D) and diabetic animals treated for 4 weeks with AG825 (1 mg/kg/ alt-diem ; +AG825). Panels b-e) are densitometry histograms showing levels of phosphorylated EGFR at the stated tyrosine residue and panel f) t-ErbB2 normalized to actin whereas panel g) shows the ratio of p-ErbB2 (Y1221/1222) to t-ErbB2. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot, Labeling, Isolation

    Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: Panel a) and c) are represenatative Western Blots following immunoprecipitations (IP) with either total-erbB2 or total-EGFR antibody and subsequent immunoblotting (IB) with both antibodies individually. Panel b) and d) represents the mean ratio of erbB2/EGFR dimers as assessed by densitometry for non-diabetic controls, (C), diabetic (D) and diabetic animals chronically treated with AG825 (+AG825) or AG1478 (+ AG1478) (both at dose of 1 mg/kg/ alt-diem ). N = 4; Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot

    A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Journal: PLoS ONE

    Article Title: Activation of ErbB2 and Downstream Signalling via Rho Kinases and ERK1/2 Contributes to Diabetes-Induced Vascular Dysfunction

    doi: 10.1371/journal.pone.0067813

    Figure Lengend Snippet: A) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in normal (5.5mM) D-glucose (NG), high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing micromolar doses of AG825 (+ AG825). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. B) Panel i) is a representative Western blot showing total (t-) or phosphorylated (p) levels of the stated proteins in VSMC grown in high glucose (25.5mM D-glucose; HG) or HG cotreated with increasing doses of anti-ErbB2 siRNA (ErbB2 siRNA) or non-targeting control siRNA (C siRNA). Panels ii-viii) are densitometry histograms showing total (t-) or phosphorylated (p-) levels of the stated proteins normalized to actin. N = 5; Mean±SD. Asterisk (*) indicates significantly different (p<0.05) mean values from normal non-diabetic rats (C) whereas hash (#) indicates significantly different mean values (p<0.05) from diabetic rats (D).

    Article Snippet: The following antibodies from Cell Signaling (USA) were used in this study: t-Her2/ErbB2-Antibody (29D8) (rabbit) Cat. No. 2165, p-Her2/ErbB2-Antibody (Tyr877) (rabbit) Cat. No. 2241, p-Her2/ErbB2-Antibody (Tyr1248; labeled in figures as Y1248a) (rabbit) Cat. No. 2247, p-Her2/ErbB2-Antibody (Tyr1248/EGFR Tyr1173; labelled as Y1248b) (rabbit) Cat. No. 2244, p-Her2/ErbB2-Antibody (Tyr1221/1222) (rabbit) Cat. No. 2243, p-ERK1/2 (p44/42 MAP Kinase, Thr202/Tyr204) Antibody (rabbit) Cat. No. 9101, : t-EGFR-Antibody (rabbit) Cat. No. 2232, p-EGFR-Antibody (Tyr1068) (rabbit) Cat. No. 2234, and p-EGFR-Antibody (Tyr1086) (rabbit).

    Techniques: Western Blot

    Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of EGFR and p-EGFR was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).

    Journal: BioMed Research International

    Article Title: Inhibition of MEIS3 Generates Cetuximab Resistance through c-Met and Akt

    doi: 10.1155/2020/2046248

    Figure Lengend Snippet: Colon cancer cell lines (a) LOVO-CON, (b) LOVO MEIS3 shRNA, (c) SNU-61 CON, and (d) SNU-61 MEIS3 were treated with different concentrations of cetuximab for 48 h. The expression of EGFR and p-EGFR was detected by western blotting ( n = 3). (e) In LOVO-CON and MEIS3 shRNA cells, expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3). (f) In SNU-61 CON and SNU-61 MEIS3 cells, the expression of MEIS3, p-EGFR, and EGFR was detected by western blot ( n = 3).

    Article Snippet: The western blot was performed in the following manner. (1) The cells were first dissolved in radioimmunoprecipitation assay (RIPA) buffer on ice for 30 min; the loading buffer was then added and boiled for 15 min. (2) The sample was loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the protein by molecular weight. (3) The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% bovine serum albumin (BSA) for two hours. (4) The membranes were labeled with the first antibody, which included the following: GAPDH (Cat No. 5174), Akt (Cat No. 4691), p-Akt (Cat No. 4060), c-Met (Cat No. 8198), phospho-c-Met (Cat No. 3077), EGFR (Cat No. 2085), and p-EGFR (Cat No. 2244), which were all purchased from Cell Signaling Technology (MA, USA).

    Techniques: shRNA, Expressing, Western Blot

    (a) Protein levels of LOVO-CON, MEIS3 shRNA, MEIS3 shRNA+c-Met overexpression, MEIS3 shRNA+c-Met shRNA, and MEIS3 overexpression+c-Met shRNA were extracted, and the expression of p-EGFR, EGFR, p-Akt, Akt, p-c-Met, and c-Met at the protein level was detected by western blot. (b) The LOVO and LOVO MEIS3 shRNA were treated with CHX for different times, and the expression of Akt was detected by western blot. (c) The SNU-61 cell line was treated with CHX for different times, and the expression of Akt was detected by western blot. (d) The LOVO cells were first treated with CHX followed by DMSO, BAF, and MG132 for different times, and the expression of Akt was determined by western blot. (e) SNU-61 (A) and HCT-116 (B) were transfected with CON and MEIS3 shRNA and then treated with DMSO, MG132, and BAF, and the degradation of Akt was determined by western blot. (f) LOVO (A) and SNU-61 (B) were transfected with the CON and MEIS3 vectors, and the ubiquitin-bonded Akt was detected using IP. (g) The LOVO cell line was transfected with random shRNA, CON vector, and c-Met shRNA for 48 h, and then, western blot was used to detect the change of c-Met in the protein level.

    Journal: BioMed Research International

    Article Title: Inhibition of MEIS3 Generates Cetuximab Resistance through c-Met and Akt

    doi: 10.1155/2020/2046248

    Figure Lengend Snippet: (a) Protein levels of LOVO-CON, MEIS3 shRNA, MEIS3 shRNA+c-Met overexpression, MEIS3 shRNA+c-Met shRNA, and MEIS3 overexpression+c-Met shRNA were extracted, and the expression of p-EGFR, EGFR, p-Akt, Akt, p-c-Met, and c-Met at the protein level was detected by western blot. (b) The LOVO and LOVO MEIS3 shRNA were treated with CHX for different times, and the expression of Akt was detected by western blot. (c) The SNU-61 cell line was treated with CHX for different times, and the expression of Akt was detected by western blot. (d) The LOVO cells were first treated with CHX followed by DMSO, BAF, and MG132 for different times, and the expression of Akt was determined by western blot. (e) SNU-61 (A) and HCT-116 (B) were transfected with CON and MEIS3 shRNA and then treated with DMSO, MG132, and BAF, and the degradation of Akt was determined by western blot. (f) LOVO (A) and SNU-61 (B) were transfected with the CON and MEIS3 vectors, and the ubiquitin-bonded Akt was detected using IP. (g) The LOVO cell line was transfected with random shRNA, CON vector, and c-Met shRNA for 48 h, and then, western blot was used to detect the change of c-Met in the protein level.

    Article Snippet: The western blot was performed in the following manner. (1) The cells were first dissolved in radioimmunoprecipitation assay (RIPA) buffer on ice for 30 min; the loading buffer was then added and boiled for 15 min. (2) The sample was loaded onto the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate the protein by molecular weight. (3) The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% bovine serum albumin (BSA) for two hours. (4) The membranes were labeled with the first antibody, which included the following: GAPDH (Cat No. 5174), Akt (Cat No. 4691), p-Akt (Cat No. 4060), c-Met (Cat No. 8198), phospho-c-Met (Cat No. 3077), EGFR (Cat No. 2085), and p-EGFR (Cat No. 2244), which were all purchased from Cell Signaling Technology (MA, USA).

    Techniques: shRNA, Over Expression, Expressing, Western Blot, Transfection, Plasmid Preparation

    (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and p-EGFR/p-HER2 levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).

    Journal: Cell reports

    Article Title: BET inhibition induces vulnerability to MCL1 targeting through upregulation of fatty acid synthesis pathway in breast cancer

    doi: 10.1016/j.celrep.2022.111304

    Figure Lengend Snippet: (A) Differential analysis of phosphoproteomic responses to BETis in resistant (HCC1954, BT474) and sensitive (SKOV3) cells. The heatmap represents the average log fold difference (log[X perturbed /X unperturbed ]) across the four BETis . “R to S deviation” is the fold changes between responses in resistant (HCC1954 and BT474) and sensitive (SKOV3) cells. The proteins with significantly different expressions between HCC1954 (most resistant) and SKOV3 (sensitive line) are listed (padj < 0.05). (B) MCL1 and signaling activity changes in response to cocktails of pathway and BET inhibitors are monitored to demonstrate the role of signaling pathways linking BET and MCL1 activity. (C) Differential analysis of transcriptomic responses (mean of duplicates) to BETi (1 μM, 24 h). The first three columns demonstrate fold changes of mRNA species that change in opposite directions in resistant versus sensitive lines. The red/white columns on right represent the involvement of each gene in different molecular processes according to a GO-term enrichment analysis. (D) Transcriptomic responses (mean of duplicates) in key genes of the lipid metabolism pathway as identified by the differential response analysis from the RNA sequencing (RNA-seq) data (left) and confirmed based on qPCR analyses for the key rate-limiting enzyme SCD. RNA-seq analysis: error bars represent the ±SEM. pPCR analysis: error bars represent the ±SD. (E) The differential analysis focusing only on total protein changes performed as in (A). The heatmaps represent the proteomic responses averaged across four BETis. (F) The analysis of SCD, MCL1, and p-EGFR/p-HER2 levels in response to inhibitors of BET (JQ1) and SCD (A939572), as well as BRD4 KDs with siRNA. (G) Representative images of IHC analysis of in vivo SCD expression in the mouse xenograft model (MDA-MB468) treated with BET and MCL1 inhibitors. Error bars represent the ±SD. (H) ChIP-seq analysis of BRD4 binding on SCD gene regulatory sites in breast cancer cell lines treated with BETis (JQ1) (see for FASN and SREBF1). (I) Correlation analysis of proteomic and transcriptomic levels involved in the associations linking BET to EGFR/HER2 within tumors of patients with ERBB2 -amplified, basal breast, and ovarian cancer. All correlations are in the range of −0.5 to 0.5. As a reference, the average correlation between mRNA and protein levels of a given gene is estimated within a range of 0.3–0.6 across the whole genome ( ; ) (data from TCGA breast and ovarian cancer projects).

    Article Snippet: p-HER2(T1248)/p-EGFR (Tyr1173) , Cell Signaling Technology , Cat# 2244, RRID:AB_331705.

    Techniques: Activity Assay, RNA Sequencing Assay, In Vivo, Expressing, ChIP-sequencing, Binding Assay, Amplification

    (A) Representative images (20×, bright field) of cellular morphology changes in response to BETi (48 h, 1 μM JQ1). (B) The BETi induces increased motility of HCC1954 (top) and no substantial difference in SKOV3 (bottom) as monitored across time points with a wound scratching assay. (C) Drug-induced changes in membrane fluidity (mean of triplicates, error bars represent ±SEM) in HCC1954 and SKOV3 cells based on fluorescent lipophilic pyrene probes that are enriched in excimer state (emission = 470 nm) with increasing membrane fluidity and enriched in monomers (emission = 400 nm) with decreasing membrane fluidity. (D) The representative images of HER2/EGFR localization changes in response to BET inhibition (48 h, 1 μM). Images are collected using fluorescent confocal microscopy (20×). The fluorescence signals from EGFR/HER2 (green) in BETi-treated HCC1954 cells suggest increased localization of the receptors. The EGFR/HER2 signal was unchanged in response to BETi in SKOV3. (E) The proposed mechanism of BET inhibition-induced vulnerability to BET and MCL1 co-targeting in the context of MCL1 chromosomal amplifications or gains based on the integrated multi-omics, network, and perturbation analyses. The marked genes and proteins are identified and validated in molecular perturbation response analyses.

    Journal: Cell reports

    Article Title: BET inhibition induces vulnerability to MCL1 targeting through upregulation of fatty acid synthesis pathway in breast cancer

    doi: 10.1016/j.celrep.2022.111304

    Figure Lengend Snippet: (A) Representative images (20×, bright field) of cellular morphology changes in response to BETi (48 h, 1 μM JQ1). (B) The BETi induces increased motility of HCC1954 (top) and no substantial difference in SKOV3 (bottom) as monitored across time points with a wound scratching assay. (C) Drug-induced changes in membrane fluidity (mean of triplicates, error bars represent ±SEM) in HCC1954 and SKOV3 cells based on fluorescent lipophilic pyrene probes that are enriched in excimer state (emission = 470 nm) with increasing membrane fluidity and enriched in monomers (emission = 400 nm) with decreasing membrane fluidity. (D) The representative images of HER2/EGFR localization changes in response to BET inhibition (48 h, 1 μM). Images are collected using fluorescent confocal microscopy (20×). The fluorescence signals from EGFR/HER2 (green) in BETi-treated HCC1954 cells suggest increased localization of the receptors. The EGFR/HER2 signal was unchanged in response to BETi in SKOV3. (E) The proposed mechanism of BET inhibition-induced vulnerability to BET and MCL1 co-targeting in the context of MCL1 chromosomal amplifications or gains based on the integrated multi-omics, network, and perturbation analyses. The marked genes and proteins are identified and validated in molecular perturbation response analyses.

    Article Snippet: p-HER2(T1248)/p-EGFR (Tyr1173) , Cell Signaling Technology , Cat# 2244, RRID:AB_331705.

    Techniques: Inhibition, Confocal Microscopy, Fluorescence

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: BET inhibition induces vulnerability to MCL1 targeting through upregulation of fatty acid synthesis pathway in breast cancer

    doi: 10.1016/j.celrep.2022.111304

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: p-HER2(T1248)/p-EGFR (Tyr1173) , Cell Signaling Technology , Cat# 2244, RRID:AB_331705.

    Techniques: Recombinant, Viability Assay, Transfection, SYBR Green Assay, Plasmid Preparation, Electron Microscopy, Expressing, Negative Control, Software, Flow Cytometry

    (A) Schematic depiction of the mutations drawn on cartoon representations of the kinase domains of HER2 and HER3. Amino acid (aa) numbering is based on the nascent reading frame. (B) CHO-K1 cells were cotransfected to overexpress HER2 and to express HER3, such as to generate constitutive HER2 autophosphorylation (lane 1) and HER3 transphosphorylation (lane 5). After 24 h, cell signaling was assayed as shown. Wild-type (WT) constructs were compared with mutants in the indicated combinations. The HER2 N-lobe mutant (I714Q) is a defective allosteric receiver, whereas the HER2 C-lobe mutant (V956R) is an impaired allosteric activator but competent receiver. HER3 is catalytically inactive and thus an obligate allosteric activator, and the HER3 C-lobe mutant (V945R) is an impaired allosteric activator. (C) CHO cells were transfected as in (B) using WT HER2 and HER3 in additional combinations. The HER2 C-lobe mutant (V956R) is an impaired allosteric activator. The HER3 single C-lobe mutant (V945R) is an impaired allosteric activator but retains some activation function. The HER3 triple C-lobe mutant (I938R-V945R-M949R) is an even more impaired allosteric activator. (D) CHO cells were transfected as in (B). The HER2 C-lobe activation function can similarly be further impaired by triple mutation. The double banding of HER3 observed in these CHO-K1 cell transfections is due to differential glycosylation . (E) CHO cells were cotransfected to overexpress HER2 and express HER3 and were treated with DMSO or either of three small molecules that bind the HER3 KD with high affinity. These were administered at 1 μM for 1 h. The binding K D s for HER3 binding are as follows: bosutinib 0.8 nM, PP-242 120 nM, and dasatinib 18 nM . The affinities for HER2 KD are >1,400 nM for these drugs. The dephosphorylation of Src is shown as a positive control for bosutinib and dasatinib, which also inhibit Src. (F) CHO-K1 cells were transfected as in (A) using WT or mutant HER2 and HER3 constructs. The HER3 K742M mutant is defective at ATP binding. Lane 8 was treated with 1 μM lapatinib for 1 h. This image was cropped down from 13 lanes for clarity. (G) CHO-K1 cells were transfected as in (C) using WT or mutant HER2 and HER3 constructs.

    Journal: Cell reports

    Article Title: Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers

    doi: 10.1016/j.celrep.2021.110291

    Figure Lengend Snippet: (A) Schematic depiction of the mutations drawn on cartoon representations of the kinase domains of HER2 and HER3. Amino acid (aa) numbering is based on the nascent reading frame. (B) CHO-K1 cells were cotransfected to overexpress HER2 and to express HER3, such as to generate constitutive HER2 autophosphorylation (lane 1) and HER3 transphosphorylation (lane 5). After 24 h, cell signaling was assayed as shown. Wild-type (WT) constructs were compared with mutants in the indicated combinations. The HER2 N-lobe mutant (I714Q) is a defective allosteric receiver, whereas the HER2 C-lobe mutant (V956R) is an impaired allosteric activator but competent receiver. HER3 is catalytically inactive and thus an obligate allosteric activator, and the HER3 C-lobe mutant (V945R) is an impaired allosteric activator. (C) CHO cells were transfected as in (B) using WT HER2 and HER3 in additional combinations. The HER2 C-lobe mutant (V956R) is an impaired allosteric activator. The HER3 single C-lobe mutant (V945R) is an impaired allosteric activator but retains some activation function. The HER3 triple C-lobe mutant (I938R-V945R-M949R) is an even more impaired allosteric activator. (D) CHO cells were transfected as in (B). The HER2 C-lobe activation function can similarly be further impaired by triple mutation. The double banding of HER3 observed in these CHO-K1 cell transfections is due to differential glycosylation . (E) CHO cells were cotransfected to overexpress HER2 and express HER3 and were treated with DMSO or either of three small molecules that bind the HER3 KD with high affinity. These were administered at 1 μM for 1 h. The binding K D s for HER3 binding are as follows: bosutinib 0.8 nM, PP-242 120 nM, and dasatinib 18 nM . The affinities for HER2 KD are >1,400 nM for these drugs. The dephosphorylation of Src is shown as a positive control for bosutinib and dasatinib, which also inhibit Src. (F) CHO-K1 cells were transfected as in (A) using WT or mutant HER2 and HER3 constructs. The HER3 K742M mutant is defective at ATP binding. Lane 8 was treated with 1 μM lapatinib for 1 h. This image was cropped down from 13 lanes for clarity. (G) CHO-K1 cells were transfected as in (C) using WT or mutant HER2 and HER3 constructs.

    Article Snippet: Anti-p-HER2 Y1248 , Cell Signaling Technology , 2247.

    Techniques: Construct, Mutagenesis, Transfection, Activation Assay, Binding Assay, De-Phosphorylation Assay, Positive Control

    (A) RMSD plot from MD simulations of the ATP-bound and apo HER3 pseudokinase states. (B) The superimposed cartoon images of the ATP-bound and apo HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (C) RMSD plot from MD simulations of the ATP-bound WT, apo WT, and apo K742M HER3 pseudokinase states. (D) The superimposed cartoon images of the ATP-bound WT, apo WT, and apo K742M HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (E) Left: plot of mutual information in kcal/mol between the transmitter (AP-2 pocket: residues 683, 685, 689, 690, 709, 718, 720, 756, and 767; side chains shown in green in right panel) and receiver (P loop: residues 696–704; side chains shown in yellow in right panel) sites, showing ~2 kcal/mol mutual information in both the absence (WT apo) and presence (WT ATP) of ATP. Error bars reflect standard deviation. Right: illustration of the normalized per-residue contribution to co-information in apo and ATP-bound simulations of HER3; “min” and “max” refer to the minimum and maximum values among all possible residues across all simulations. Residues colored dark red contribute most to communication between the AP-2 pocket and the P loop. (F) CHO-K1 cells were transfected to overexpress HER2 and to express the indicated WT or mutant HER3 constructs. The surface residues F704, L709, and V786 were mutated to impair the binding affinity of the AP-2 pocket. (G) W728 was mutated to collapse the AP-2 pocket. (H) The effect of ligand stimulation was studied on the F704 AP-2 pocket mutant.

    Journal: Cell reports

    Article Title: Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers

    doi: 10.1016/j.celrep.2021.110291

    Figure Lengend Snippet: (A) RMSD plot from MD simulations of the ATP-bound and apo HER3 pseudokinase states. (B) The superimposed cartoon images of the ATP-bound and apo HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (C) RMSD plot from MD simulations of the ATP-bound WT, apo WT, and apo K742M HER3 pseudokinase states. (D) The superimposed cartoon images of the ATP-bound WT, apo WT, and apo K742M HER3 final pseudokinase states showcase inward movement of the β3-β4 loop and the collapse of the P loop in the apo state. (E) Left: plot of mutual information in kcal/mol between the transmitter (AP-2 pocket: residues 683, 685, 689, 690, 709, 718, 720, 756, and 767; side chains shown in green in right panel) and receiver (P loop: residues 696–704; side chains shown in yellow in right panel) sites, showing ~2 kcal/mol mutual information in both the absence (WT apo) and presence (WT ATP) of ATP. Error bars reflect standard deviation. Right: illustration of the normalized per-residue contribution to co-information in apo and ATP-bound simulations of HER3; “min” and “max” refer to the minimum and maximum values among all possible residues across all simulations. Residues colored dark red contribute most to communication between the AP-2 pocket and the P loop. (F) CHO-K1 cells were transfected to overexpress HER2 and to express the indicated WT or mutant HER3 constructs. The surface residues F704, L709, and V786 were mutated to impair the binding affinity of the AP-2 pocket. (G) W728 was mutated to collapse the AP-2 pocket. (H) The effect of ligand stimulation was studied on the F704 AP-2 pocket mutant.

    Article Snippet: Anti-p-HER2 Y1248 , Cell Signaling Technology , 2247.

    Techniques: Standard Deviation, Transfection, Mutagenesis, Construct, Binding Assay

    (A) HCC1569 human HER2-amplified breast cancer cells were engineered to eliminate HER3 expression by CRISPR-Cas targeting (HCC1569-HER3KO). To eliminate the role of clonal growth characteristics in the replacement experiments, we mixed together three separate clones of HCC1569-HER3KO cells to generate a polyclonal HCC1569-HER3KO cell line (lane 2), and this cell line was used as the parental cell line for the various add-back experiments. These were then transduced to re-express WT HER3 (lane 4) or experimental mutant HER3 constructs. Experimental add-backs included a HER3 C-lobe mutant defective at allosteric activation (HER3 I938R/V945R/M949R) and a HER3 with a mutated AP-2 pocket (F704D). The expression of firefly luciferase (lane 3) constitutes a negative control cell type. The add-back HER3 constructs contain C-terminal myc tags. (B) The indicated engineered versions of HER2-amplified HCC1569 tumor cells were inoculated subcutaneously in NSG mice, and tumor volumes were measured over time. The number of surviving mice along the time course of the animal studies is shown for each arm underneath, and the sample size reduction over time in some arms reflects the removal of mice for euthanasia because of large tumor sizes as mandated by guidelines. The error bars reflect SEM.

    Journal: Cell reports

    Article Title: Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers

    doi: 10.1016/j.celrep.2021.110291

    Figure Lengend Snippet: (A) HCC1569 human HER2-amplified breast cancer cells were engineered to eliminate HER3 expression by CRISPR-Cas targeting (HCC1569-HER3KO). To eliminate the role of clonal growth characteristics in the replacement experiments, we mixed together three separate clones of HCC1569-HER3KO cells to generate a polyclonal HCC1569-HER3KO cell line (lane 2), and this cell line was used as the parental cell line for the various add-back experiments. These were then transduced to re-express WT HER3 (lane 4) or experimental mutant HER3 constructs. Experimental add-backs included a HER3 C-lobe mutant defective at allosteric activation (HER3 I938R/V945R/M949R) and a HER3 with a mutated AP-2 pocket (F704D). The expression of firefly luciferase (lane 3) constitutes a negative control cell type. The add-back HER3 constructs contain C-terminal myc tags. (B) The indicated engineered versions of HER2-amplified HCC1569 tumor cells were inoculated subcutaneously in NSG mice, and tumor volumes were measured over time. The number of surviving mice along the time course of the animal studies is shown for each arm underneath, and the sample size reduction over time in some arms reflects the removal of mice for euthanasia because of large tumor sizes as mandated by guidelines. The error bars reflect SEM.

    Article Snippet: Anti-p-HER2 Y1248 , Cell Signaling Technology , 2247.

    Techniques: Amplification, Expressing, CRISPR, Clone Assay, Mutagenesis, Construct, Activation Assay, Luciferase, Negative Control

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Targetable HER3 functions driving tumorigenic signaling in HER2-amplified cancers

    doi: 10.1016/j.celrep.2021.110291

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Anti-p-HER2 Y1248 , Cell Signaling Technology , 2247.

    Techniques: Recombinant, Transfection, Plasmid Preparation, BIA-KA, Mutagenesis, Modification, Software, Expressing