Santa Cruz Biotechnology sgk269

Figure 1. Phosphoproteomic analysis of basal breast cancer cell lines following Lyn knockdown. A, phosphoproteomic proﬁling workﬂow. B, validation of Lyn knockdown by Western blotting. C, Venn diagram displaying proteins identiﬁed as Lyn substrates in BT-549 and MDA-MB-231 cells. D, biochemical conﬁrmation of Lyn-dependent phosphorylation of <t>SgK269</t> on Y635. SgK269 immunoprecipitates were Western blotted using a pY635 phosphospeciﬁc or total SgK269 antibody. Cell lysates were blotted as indicated. Grb2 served as a loading control.

Sgk269, supplied by Santa Cruz Biotechnology, 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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Journal: Cancer Research

Article Title: Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

doi: 10.1158/0008-5472.can-12-1472

Figure Lengend Snippet: Figure 1. Phosphoproteomic analysis of basal breast cancer cell lines following Lyn knockdown. A, phosphoproteomic proﬁling workﬂow. B, validation of Lyn knockdown by Western blotting. C, Venn diagram displaying proteins identiﬁed as Lyn substrates in BT-549 and MDA-MB-231 cells. D, biochemical conﬁrmation of Lyn-dependent phosphorylation of SgK269 on Y635. SgK269 immunoprecipitates were Western blotted using a pY635 phosphospeciﬁc or total SgK269 antibody. Cell lysates were blotted as indicated. Grb2 served as a loading control.

Article Snippet: All antibodies were from Cell Signaling Technology, except: PY20 (Abcam); SgK269 and Tubulin (Santa Cruz Biotechnology); b-actin (Sigma); Shc, Grb2, and E-cadherin (BD Biosciences); HA (Roche); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Ambion).

Techniques: Knockdown, Biomarker Discovery, Western Blot, Phospho-proteomics, Control

Figure 2. SgK269 protein expression is elevated in basal breast cancer cell lines and a subset of primary breast cancers. A, Western blot analysis of SgK269 and Lyn expression across a panel of breast cancer cell lines. B, SgK269 mRNA expression across breast cancer cell lines. Data are expressed relative to the expression level of MCF-7 cells (mean SEM, n ¼ 3). C, Western blot analysis of SgK269 expression in different subtypes of primary breast cancer.

Journal: Cancer Research

Article Title: Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

doi: 10.1158/0008-5472.can-12-1472

Figure Lengend Snippet: Figure 2. SgK269 protein expression is elevated in basal breast cancer cell lines and a subset of primary breast cancers. A, Western blot analysis of SgK269 and Lyn expression across a panel of breast cancer cell lines. B, SgK269 mRNA expression across breast cancer cell lines. Data are expressed relative to the expression level of MCF-7 cells (mean SEM, n ¼ 3). C, Western blot analysis of SgK269 expression in different subtypes of primary breast cancer.

Techniques: Expressing, Western Blot

Figure 3. SgK269 promotes EMT in MCF-10A cells. A, SgK269 was stably expressed in MCF-10A cells and cell lysates blotted for the indicated proteins. Left, lysate from MDA-MB-231 basal breast cancer cells was included for comparison. B, photomicrographs of MCF-10A cells expressing SgK269 and vector control cells (scale bar, 50 mm). The histogram on the left indicates mean cell length from the 2 cell populations (mean SEM, n ¼ 100; , P < 0.05). C, changes in expression of speciﬁc EMT markers induced by SgK269. Expression of 84 EMT-related genes was quantiﬁed by qRT-PCR and genes exhibiting signiﬁcant (P < 0.05) changes in replicate experiments selected. The graphs indicate fold expression changes in genes normally upregulated (top) or downregulated (bottom) during EMT. D, random cell motility of MCF-10A cells expressing SgK269, as determined by live cell tracking (mean SEM, n ¼ 50; , P < 0.05). E, ﬂuorescence microscopy of MCF-10A cells expressing SgK269, stained with an anti-HA antibody and phalloidin. Scale bar, 10 mm.

Journal: Cancer Research

Article Title: Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

doi: 10.1158/0008-5472.can-12-1472

Figure Lengend Snippet: Figure 3. SgK269 promotes EMT in MCF-10A cells. A, SgK269 was stably expressed in MCF-10A cells and cell lysates blotted for the indicated proteins. Left, lysate from MDA-MB-231 basal breast cancer cells was included for comparison. B, photomicrographs of MCF-10A cells expressing SgK269 and vector control cells (scale bar, 50 mm). The histogram on the left indicates mean cell length from the 2 cell populations (mean SEM, n ¼ 100; , P < 0.05). C, changes in expression of speciﬁc EMT markers induced by SgK269. Expression of 84 EMT-related genes was quantiﬁed by qRT-PCR and genes exhibiting signiﬁcant (P < 0.05) changes in replicate experiments selected. The graphs indicate fold expression changes in genes normally upregulated (top) or downregulated (bottom) during EMT. D, random cell motility of MCF-10A cells expressing SgK269, as determined by live cell tracking (mean SEM, n ¼ 50; , P < 0.05). E, ﬂuorescence microscopy of MCF-10A cells expressing SgK269, stained with an anti-HA antibody and phalloidin. Scale bar, 10 mm.

Techniques: Stable Transfection, Comparison, Expressing, Plasmid Preparation, Control, Quantitative RT-PCR, Cell Tracking Assay, Microscopy, Staining

Figure 6. Determination of protein–protein interactions and signaling pathways regulated by Y635 phosphorylation. A, coimmunoprecipitation studies with WT SgK269. B, coimmunoprecipitation of Grb2 and Shc with WT and Y635F SgK269. The histogram provides quantiﬁcation of the Grb2 interaction with Y635F SgK269 normalized to the respective interaction with WT SgK269. Data represent mean SEM, n ¼ 5. , P < 0.05. C, effect of the SFK inhibitor PP2 on SgK269/Grb2 interaction. SgK269 immunoprecipitates were Western blotted as indicated. For the experiment from MDA-MB-231 cells, separate immunoprecipitates were Western blotted for pY635 and Grb2, and the loading controls are provided below and above the pY635 and Grb2 blots, respectively. D, effect of Lyn knockdown on SgK269/Grb2 interaction. SgK269 immunoprecipitates and corresponding cell lysates were Western blotted as indicated. E, phosphorylated Y635 represents a binding site for the Grb2 SH2 domain. Left, GST or GST-Grb2 SH2 immobilized on beads was used in pull-down assays from the indicated cell lysates. Bound SgK269 was detected by Western blotting and equal loading conﬁrmed by gel staining with Coomassie Blue. Right, pull-down assays were undertaken in the presence or absence of phosphorylated or nonphosphorylated Y635 peptides.

Journal: Cancer Research

Article Title: Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

doi: 10.1158/0008-5472.can-12-1472

Figure Lengend Snippet: Figure 6. Determination of protein–protein interactions and signaling pathways regulated by Y635 phosphorylation. A, coimmunoprecipitation studies with WT SgK269. B, coimmunoprecipitation of Grb2 and Shc with WT and Y635F SgK269. The histogram provides quantiﬁcation of the Grb2 interaction with Y635F SgK269 normalized to the respective interaction with WT SgK269. Data represent mean SEM, n ¼ 5. , P < 0.05. C, effect of the SFK inhibitor PP2 on SgK269/Grb2 interaction. SgK269 immunoprecipitates were Western blotted as indicated. For the experiment from MDA-MB-231 cells, separate immunoprecipitates were Western blotted for pY635 and Grb2, and the loading controls are provided below and above the pY635 and Grb2 blots, respectively. D, effect of Lyn knockdown on SgK269/Grb2 interaction. SgK269 immunoprecipitates and corresponding cell lysates were Western blotted as indicated. E, phosphorylated Y635 represents a binding site for the Grb2 SH2 domain. Left, GST or GST-Grb2 SH2 immobilized on beads was used in pull-down assays from the indicated cell lysates. Bound SgK269 was detected by Western blotting and equal loading conﬁrmed by gel staining with Coomassie Blue. Right, pull-down assays were undertaken in the presence or absence of phosphorylated or nonphosphorylated Y635 peptides.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot, Knockdown, Binding Assay, Staining

Figure 7. Functional analysis of SgK269 in MCF-10A and basal breast cancer cells. A, signaling pathway activation in MCF-10A cells. Cell lysates from monolayers or 3D cultures grown for 4 days were Western blotted as indicated. Numbers below the panels indicate normalized (relative to total) levels of pStat3, pErk, and pAkt, expressed relative to the value for vector controls, which is arbitrarily set at 1.0. B, MEK inhibition reverses the effect of SgK269 on acinar size. Top left, Western blotting on acinar lysates grown for 4 days in the presence of PD184352 at the indicated concentrations. Right, photomicrographs and bottom left, acinar size quantiﬁcation, of the corresponding acini grown in Matrigel for 12 days. Data represent mean SEM, n ¼ 100. , P < 0.05 relative to vector control acini. Scale bar, 100 mm. C, stable SgK269 knockdown in MDA-MB-231 cells induces mesenchymal-to-epithelial transition. Left, lysates from control and SgK269 knockdown cells were Western blotted as indicated. Right, morphology of the corresponding cell pools, as indicated by light microscopy. Scale bar, 50 mm. D, effect of stable SgK269 knockdown in BT-549 and MDA-MB-468 basal breast cancer cells. Cell lysates were Western blotted with the indicated antibodies. The histogram provides the results for anchorage-independent growth assays where control or SgK269 knockdown BT549 cells were grown in soft agar for 14 days. Data represent mean SEM, n ¼ 3. , P < 0.05.

Journal: Cancer Research

Article Title: Involvement of Lyn and the Atypical Kinase SgK269/PEAK1 in a Basal Breast Cancer Signaling Pathway

doi: 10.1158/0008-5472.can-12-1472

Figure Lengend Snippet: Figure 7. Functional analysis of SgK269 in MCF-10A and basal breast cancer cells. A, signaling pathway activation in MCF-10A cells. Cell lysates from monolayers or 3D cultures grown for 4 days were Western blotted as indicated. Numbers below the panels indicate normalized (relative to total) levels of pStat3, pErk, and pAkt, expressed relative to the value for vector controls, which is arbitrarily set at 1.0. B, MEK inhibition reverses the effect of SgK269 on acinar size. Top left, Western blotting on acinar lysates grown for 4 days in the presence of PD184352 at the indicated concentrations. Right, photomicrographs and bottom left, acinar size quantiﬁcation, of the corresponding acini grown in Matrigel for 12 days. Data represent mean SEM, n ¼ 100. , P < 0.05 relative to vector control acini. Scale bar, 100 mm. C, stable SgK269 knockdown in MDA-MB-231 cells induces mesenchymal-to-epithelial transition. Left, lysates from control and SgK269 knockdown cells were Western blotted as indicated. Right, morphology of the corresponding cell pools, as indicated by light microscopy. Scale bar, 50 mm. D, effect of stable SgK269 knockdown in BT-549 and MDA-MB-468 basal breast cancer cells. Cell lysates were Western blotted with the indicated antibodies. The histogram provides the results for anchorage-independent growth assays where control or SgK269 knockdown BT549 cells were grown in soft agar for 14 days. Data represent mean SEM, n ¼ 3. , P < 0.05.

Techniques: Functional Assay, Activation Assay, Western Blot, Plasmid Preparation, Inhibition, Control, Knockdown, Light Microscopy

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