p bcr abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p bcr abl y412
    HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and <t>BCR-ABL/PI3K/Akt</t> in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.
    P Bcr Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Images

    1) Product Images from "Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1"

    Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2022.023

    HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.
    Figure Legend Snippet: HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Quantitative RT-PCR, Western Blot

    HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.
    Figure Legend Snippet: HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.

    Techniques Used: Transfection, Plasmid Preparation, Positive Control, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    p bcr abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p bcr abl y412
    HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and <t>BCR-ABL/PI3K/Akt</t> in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.
    P Bcr Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p bcr abl y412/product/Cell Signaling Technology Inc
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p bcr abl y412 - by Bioz Stars, 2023-03
    93/100 stars

    Images

    1) Product Images from "Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1"

    Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

    Journal: Biomolecules & Therapeutics

    doi: 10.4062/biomolther.2022.023

    HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.
    Figure Legend Snippet: HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Quantitative RT-PCR, Western Blot

    HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.
    Figure Legend Snippet: HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.

    Techniques Used: Transfection, Plasmid Preparation, Positive Control, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    c abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc c abl y412
    Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” <t>(Y412</t> and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).
    C Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair"

    Article Title: c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21197277

    Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” (Y412 and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).
    Figure Legend Snippet: Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” (Y412 and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).

    Techniques Used: Activity Assay, Generated, Irradiation, Western Blot, Expressing

    c abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc c abl y412
    C Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    c abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc c abl y412
    C Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c abl y412/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    bsa cst 3098 p c abl y412 rabbit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc bsa cst 3098 p c abl y412 rabbit
    List of Primary Antibodies Used in the Study
    Bsa Cst 3098 P C Abl Y412 Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 1 article reviews
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    Images

    1) Product Images from "Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration"

    Article Title: Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2017.06.009

    List of Primary Antibodies Used in the Study
    Figure Legend Snippet: List of Primary Antibodies Used in the Study

    Techniques Used: Blocking Assay

    Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.
    Figure Legend Snippet: Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.

    Techniques Used: Western Blot, Positive Control, Expressing, Molecular Weight, Immunoprecipitation, Binding Assay, Staining

    bsa cst 3098 p c abl y412 rabbit  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc bsa cst 3098 p c abl y412 rabbit
    List of Primary Antibodies Used in the Study
    Bsa Cst 3098 P C Abl Y412 Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsa cst 3098 p c abl y412 rabbit/product/Cell Signaling Technology Inc
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    bsa cst 3098 p c abl y412 rabbit - by Bioz Stars, 2023-03
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    Images

    1) Product Images from "Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration"

    Article Title: Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2017.06.009

    List of Primary Antibodies Used in the Study
    Figure Legend Snippet: List of Primary Antibodies Used in the Study

    Techniques Used: Blocking Assay

    Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.
    Figure Legend Snippet: Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.

    Techniques Used: Western Blot, Positive Control, Expressing, Molecular Weight, Immunoprecipitation, Binding Assay, Staining

    p c abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p c abl y412
    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased <t>p-c-ABL(Y412),</t> p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.
    P C Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ABL regulation by AXL promotes cisplatin resistance in esophageal cancer"

    Article Title: ABL regulation by AXL promotes cisplatin resistance in esophageal cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-12-3151

    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased p-c-ABL(Y412), p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.
    Figure Legend Snippet: A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased p-c-ABL(Y412), p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.

    Techniques Used: Stable Transfection, Expressing, Western Blot, Immunoprecipitation, Transfection

    p c abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc p c abl y412
    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased <t>p-c-ABL(Y412),</t> p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.
    P C Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ABL regulation by AXL promotes cisplatin resistance in esophageal cancer"

    Article Title: ABL regulation by AXL promotes cisplatin resistance in esophageal cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-12-3151

    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased p-c-ABL(Y412), p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.
    Figure Legend Snippet: A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased p-c-ABL(Y412), p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.

    Techniques Used: Stable Transfection, Expressing, Western Blot, Immunoprecipitation, Transfection

    p c abl y412 rabbit polyclonal antibody  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc p c abl y412 rabbit polyclonal antibody
    P C Abl Y412 Rabbit Polyclonal 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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    c abl y412  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc c abl y412
    C Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p bcr abl y412
    HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and <t>BCR-ABL/PI3K/Akt</t> in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.
    P Bcr Abl Y412, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc c abl y412
    Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” <t>(Y412</t> and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).
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    Cell Signaling Technology Inc bsa cst 3098 p c abl y412 rabbit
    List of Primary Antibodies Used in the Study
    Bsa Cst 3098 P C Abl Y412 Rabbit, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p c abl y412
    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased <t>p-c-ABL(Y412),</t> p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.
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    Cell Signaling Technology Inc p c abl y412 rabbit polyclonal antibody
    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased <t>p-c-ABL(Y412),</t> p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.
    P C Abl Y412 Rabbit Polyclonal 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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    Image Search Results


    HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.

    Journal: Biomolecules & Therapeutics

    Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

    doi: 10.4062/biomolther.2022.023

    Figure Lengend Snippet: HST regulates the sphingolipid rheostat, represses SPHK1/S1P/S1PR1 and BCR-ABL/PI3K/Akt in CML cells. (A, B) K562 cells and K562/G01 cells were exposed with HST for 48 h. Cer concentration (A) and extracellular S1P level (B) were detected by ELISA. (C) SPHK1 activity was detected by ELISA. (D) S1PR1 level was analyzed by qRT-PCR. (E) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. Data represent mean value ± SD. * p <0.05, ** p <0.01, *** p <0.001.

    Article Snippet: Antibodies against Cyclin B1 (1:1,000, #4135), CDC2 (1:1,000, #9116), Cyclin D1 (1:1,000, #55506), Caspase 3 (1:1,000, #9662), PARP (1:1,000, #9532), Caspase-8 (1:1,000, #9746), Caspase-9 (1:1,000, #9502), Cytochrome c (1:1,000, #4272), BCR-ABL (1:1,000, #2862), p-BCR-ABL (Y412) (1:1,000, #2865), PI3K-p110α (1:1,000, #4249), p-Akt (Ser473) (1:1,000, #3787), Akt (1:1,000, #9272), β-actin (1:1,000, #8457), anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (1:2,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Quantitative RT-PCR, Western Blot

    HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.

    Journal: Biomolecules & Therapeutics

    Article Title: Growth Inhibitory and Pro-Apoptotic Effects of Hirsuteine in Chronic Myeloid Leukemia Cells through Targeting Sphingosine Kinase 1

    doi: 10.4062/biomolther.2022.023

    Figure Lengend Snippet: HST exerts anti-leukemic effect in CML cells through SPHK1 suppression. K562 cells and K562/G01 cells were exposed with HST for 48 h after transfected with SPHK1 vector or empty vector for 6 h. PF543 (30 μM in K562, 40 μM in K562/G01) was used as a positive control. (A) SPHK1 expression was detected by western blot. (B, C) Cell viability was conducted using MTT. (D-F) Cell apoptosis was analyzed using flow cytometry. (G, H) Extracellular S1P level (G) and Cer concentration (H) were detected by ELISA. (I) SPHK1 and BCR-ABL/PI3K/Akt signaling were detected by western blot. (J) S1PR1 level was determined using qRT-PCR. Data represent mean value ± SD. * p <0.05, *** p <0.001 versus the empty vector-transfected cells; ## p <0.01, ### p <0.001 versus the empty vector-transfected cells treated with HST.

    Article Snippet: Antibodies against Cyclin B1 (1:1,000, #4135), CDC2 (1:1,000, #9116), Cyclin D1 (1:1,000, #55506), Caspase 3 (1:1,000, #9662), PARP (1:1,000, #9532), Caspase-8 (1:1,000, #9746), Caspase-9 (1:1,000, #9502), Cytochrome c (1:1,000, #4272), BCR-ABL (1:1,000, #2862), p-BCR-ABL (Y412) (1:1,000, #2865), PI3K-p110α (1:1,000, #4249), p-Akt (Ser473) (1:1,000, #3787), Akt (1:1,000, #9272), β-actin (1:1,000, #8457), anti-rabbit and anti-mouse HRP-conjugated secondary antibodies (1:2,000) were purchased from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Transfection, Plasmid Preparation, Positive Control, Expressing, Western Blot, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

    Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” (Y412 and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).

    Journal: International Journal of Molecular Sciences

    Article Title: c-Abl Tyrosine Kinase Is Regulated Downstream of the Cytoskeletal Protein Synemin in Head and Neck Squamous Cell Carcinoma Radioresistance and DNA Repair

    doi: 10.3390/ijms21197277

    Figure Lengend Snippet: Synemin regulates c-Abl kinase activity. ( A ) Heatmap of tyrosine kinase activities in cells depleted of synemin and exposed to 6 Gy X-ray. Kinase activity profiles were generated by PamGene Technology. ( B ) Normalized kinase statistic of un-irradiated controls (grey) and samples 1 h (yellow) or 24 h (orange) post 6 Gy X-ray exposure. ( C ) Top affected tyrosine kinases in synemin-depleted cell cultures 24 h post 6 Gy X-ray irradiation. X -axis indicates the τ value for each kinase (τ < 0 indicates reduced kinase activity relative to control). Dot colors indicate the specificity score. Dot size indicates the number of included peptides. The Y -axis shows the overall score. ( D ) Western blotting of c-Abl expression and phosphorylation from whole cell lysates of synemin-depleted cells. ( E ) Densitometries of Western blots from synemin-depleted, 6-Gy irradiated cells showing phosphorylated forms of c-Abl shown in “D” (Y412 and T735, n = 4). Phosphorylation levels were calculated relative to the total amount of c-Abl. Dots represent each independent experiment. Data are presented as mean ± SD (two-sided t -test; * p < 0.05).

    Article Snippet: Antibodies against c-Abl (#2862P), c-Abl T715 (#2846S), and c-Abl Y412 (#2865S) were from Cell Signaling (Frankfurt, Germany); β-actin (#A1978) and Synemin (#S9075, for zebrafish western blot) were from Sigma-Aldrich (Taufkirchen, Germany); γH2AX S139 (#05-636) was from Upstate (Schwalbach, Germany) and 53BP1 (#NB100-904) was from Novus Biologicals (Cambridge, UK).

    Techniques: Activity Assay, Generated, Irradiation, Western Blot, Expressing

    List of Primary Antibodies Used in the Study

    Journal: The American Journal of Pathology

    Article Title: Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration

    doi: 10.1016/j.ajpath.2017.06.009

    Figure Lengend Snippet: List of Primary Antibodies Used in the Study

    Article Snippet: For detection of PDGFs in HHSteC media, media were concentrated after 24 or 48 hours of serum starvation using Amicon Ultra-15 Centrifugal Filters (Millipore) and resuspended in sample buffer to perform western blot analysis, as described earlier in this paragraph. table ft1 table-wrap mode="anchored" t5 caption a7 Primary antibody Species Dilution Block Company Catalog no. Akt Rabbit 1:1000 5% BSA CST (Danvers, MA) 4685 P-S473-Akt Rabbit 1:1000 5% BSA CST 4060 c-Abl Rabbit 1:1000 5% BSA CST 2862 P-Y89-c-Abl Rabbit 1:1000 5% BSA CST 3098 P-c-Abl Y412 Rabbit 1:1000 5% BSA CST 2865 CrkII Rabbit 1:1000 5% BSA CST 3492 P-Y221-CrkII Mouse 1:1000 5% Milk CST 3491 CrkL Rabbit 1:1000 5% BSA CST 3182 P-Y207-CrkL Rabbit 1:1000 5% BSA CST 3181 Elk-1 Rabbit 1:1000 5% BSA CST 9182 P-S383 Elk-1 Rabbit 1:1000 5% BSA CST 9181 Erk1/2 Rabbit 1:1000 5% BSA CST 4695 P-T202/Y204-Erk1/2 Rabbit 1:1000 5% BSA CST 4370 FAK Rabbit 1:1000 5% BSA CST 13009 P-Y397-FAK Rabbit 1:1000 5% BSA CST 8556 P-Y576/577-FAK Rabbit 1:1000 5% BSA CST 3281 P-Y925-FAK Rabbit 1:1000 5% BSA CST 3284 P-S2448-mTOR Rabbit 1:500 5% BSA CST 2971 P-S2481-mTOR Rabbit 1:1000 5% BSA CST 2974 p38 Rabbit 1:1000 5% BSA CST 8690 P-T180/Y182-p38 Rabbit 1:200 5% Milk CST 4511 PDGFA Mouse 1:200 5% Milk SCBT (Dallas, TX) sc-9974 PDGFB Rabbit 1:1000 5% Milk Abcam (Cambridge, UK) ab23914 PDGFC Goat 1:200 3% BSA R&D Systems (Minneapolis, MN) AF1560 PDGFD Goat 1:1000 5% BSA R&D Systems AF1159 PDGFRα Rabbit 1:300 5% Milk SCBT sc-338 PDGFRβ Rabbit 1:1000 5% BSA SCBT sc-432 P-Y572/574-PDGFRα/β Rabbit 1:1000 5% BSA Invitrogen (Carlsbad, CA) 44-1000G P-Y742-PDGFRα Rabbit 1:1000 5% BSA Invitrogen 44-1006 P-Y762-PDGFRα Rabbit 1:1000 5% BSA CST 24188 P-Y849/βY857-PDGFRα Rabbit 1:1000 5% BSA CST 3170 P-Y1018-PDGFRα Rabbit 1:1000 5% BSA CST 4547 Pan-phospho-PKC Rabbit 1:1000 5% BSA CST 9371 Open in a separate window BSA, bovine serum albumin; Crk, C10 regulator of kinase; CrkL, Crk-like; CST, Cell Signaling Technology; Erk1/2, extracellular signal–regulated kinase 1 and 2; FAK, focal adhesion kinase; mTOR, mechanistic target of rapamycin; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PKC, protein kinase C; SCBT, Santa Cruz Biotechnology.

    Techniques: Blocking Assay

    Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.

    Journal: The American Journal of Pathology

    Article Title: Platelet-Derived Growth Factor Receptor α Contributes to Human Hepatic Stellate Cell Proliferation and Migration

    doi: 10.1016/j.ajpath.2017.06.009

    Figure Lengend Snippet: Olaratumab inhibits baseline PDGFRα signaling in HHSteCs along with downstream effectors. A: Representative Western blots from HHSteC treatment with 300 nmol/L olaratumab using pooled lysates from three technical replicates for each time point showing decreased PDGFRα phosphorylation at Y762 and Y849 compared to IgG-treated controls. PDGF-BB treatment included as a positive control. B: Similar representative Western blots showing decreased Erk and Elk-1 phosphorylation compared to IgG-treated controls. PDGF-BB treatment serves as a positive control. C: Representative Western blots show olaratumab treatment decreases phosphorylation of mTOR at Ser2448 and increases phosphorylation at Ser2481. Olaratumab also decreases p38 phosphorylation. D: Similar representative Western blots showing olaratumab treatment increasing Abl expression and phosphorylation at Y412 and Y89 and increasing phosphorylation at inhibitory tyrosine residues of CrkII (Y221) and CrkL (Y207). Arrowhead indicates the correct molecular weight band. E: Immunoprecipitation of HHSteC lysates using anti-CrkII shows increased binding of CrkII to both total PDGFRα and phospho-PDGFRα Y762 after olaratumab treatment. Ponceau staining is included as a loading control. All Western blots were repeated twice on the same pooled lysates from three technical replicates and performed for the two stellate cell batches. h, hours; IP, immunoprecipitation; m, minutes; WB, Western blotting.

    Article Snippet: For detection of PDGFs in HHSteC media, media were concentrated after 24 or 48 hours of serum starvation using Amicon Ultra-15 Centrifugal Filters (Millipore) and resuspended in sample buffer to perform western blot analysis, as described earlier in this paragraph. table ft1 table-wrap mode="anchored" t5 caption a7 Primary antibody Species Dilution Block Company Catalog no. Akt Rabbit 1:1000 5% BSA CST (Danvers, MA) 4685 P-S473-Akt Rabbit 1:1000 5% BSA CST 4060 c-Abl Rabbit 1:1000 5% BSA CST 2862 P-Y89-c-Abl Rabbit 1:1000 5% BSA CST 3098 P-c-Abl Y412 Rabbit 1:1000 5% BSA CST 2865 CrkII Rabbit 1:1000 5% BSA CST 3492 P-Y221-CrkII Mouse 1:1000 5% Milk CST 3491 CrkL Rabbit 1:1000 5% BSA CST 3182 P-Y207-CrkL Rabbit 1:1000 5% BSA CST 3181 Elk-1 Rabbit 1:1000 5% BSA CST 9182 P-S383 Elk-1 Rabbit 1:1000 5% BSA CST 9181 Erk1/2 Rabbit 1:1000 5% BSA CST 4695 P-T202/Y204-Erk1/2 Rabbit 1:1000 5% BSA CST 4370 FAK Rabbit 1:1000 5% BSA CST 13009 P-Y397-FAK Rabbit 1:1000 5% BSA CST 8556 P-Y576/577-FAK Rabbit 1:1000 5% BSA CST 3281 P-Y925-FAK Rabbit 1:1000 5% BSA CST 3284 P-S2448-mTOR Rabbit 1:500 5% BSA CST 2971 P-S2481-mTOR Rabbit 1:1000 5% BSA CST 2974 p38 Rabbit 1:1000 5% BSA CST 8690 P-T180/Y182-p38 Rabbit 1:200 5% Milk CST 4511 PDGFA Mouse 1:200 5% Milk SCBT (Dallas, TX) sc-9974 PDGFB Rabbit 1:1000 5% Milk Abcam (Cambridge, UK) ab23914 PDGFC Goat 1:200 3% BSA R&D Systems (Minneapolis, MN) AF1560 PDGFD Goat 1:1000 5% BSA R&D Systems AF1159 PDGFRα Rabbit 1:300 5% Milk SCBT sc-338 PDGFRβ Rabbit 1:1000 5% BSA SCBT sc-432 P-Y572/574-PDGFRα/β Rabbit 1:1000 5% BSA Invitrogen (Carlsbad, CA) 44-1000G P-Y742-PDGFRα Rabbit 1:1000 5% BSA Invitrogen 44-1006 P-Y762-PDGFRα Rabbit 1:1000 5% BSA CST 24188 P-Y849/βY857-PDGFRα Rabbit 1:1000 5% BSA CST 3170 P-Y1018-PDGFRα Rabbit 1:1000 5% BSA CST 4547 Pan-phospho-PKC Rabbit 1:1000 5% BSA CST 9371 Open in a separate window BSA, bovine serum albumin; Crk, C10 regulator of kinase; CrkL, Crk-like; CST, Cell Signaling Technology; Erk1/2, extracellular signal–regulated kinase 1 and 2; FAK, focal adhesion kinase; mTOR, mechanistic target of rapamycin; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PKC, protein kinase C; SCBT, Santa Cruz Biotechnology.

    Techniques: Western Blot, Positive Control, Expressing, Molecular Weight, Immunoprecipitation, Binding Assay, Staining

    A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased p-c-ABL(Y412), p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.

    Journal: Cancer research

    Article Title: ABL regulation by AXL promotes cisplatin resistance in esophageal cancer

    doi: 10.1158/0008-5472.CAN-12-3151

    Figure Lengend Snippet: A) OE33 cells stably expressing AXL or pcDNA4 were treated with vehicle or CDDP (10 μmol/L) for 48h. The Western blot data indicate that CDDP treatment significantly increased p-c-ABL(Y412), p-p73β(Y99), and p73β protein levels in control cells. In contrast, AXL expression abrogated these effects in response to CDDP. B) Western blot analysis of immunoprecipitated exogenous p73β protein with M2-flag antibody in OE33 cells transfected with p73β-Flag alone or in combination with AXL. Exogenous p73β interacted with endogenous c-ABL protein, and the p73β/c-ABL protein complex was disrupted by AXL. C) Cell viability of OE33 cells transfected with pcDNA4 alone, AXL alone or in combination with GFP-c-ABL was assessed 48h post-transfection. AXL expression blocked c-ABL-induced cell death. D) Western blot analysis of c-ABL and AXL proteins in OE33 cells transiently transfected as in panel C.

    Article Snippet: AXL, PUMA, c-ABL, p-c-ABL(Y412), cleaved caspase-3 and -9, cleaved PARP, and β-actin antibodies were obtained from Cell Signaling Technology (Danvers, MA). p-AXL(Y779) and HDM2 antibodies were purchased from R&D Systems (Minneapolis, MN) and Calbiochem (Billerica, MA), respectively. p73 antibody was obtained from Bethyl Laboratories (Montgomery, TX).

    Techniques: Stable Transfection, Expressing, Western Blot, Immunoprecipitation, Transfection