Journal: The American Journal of Pathology

Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

doi: 10.1016/j.ajpath.2019.10.017

Figure Lengend Snippet: Antibodies and Dilutions Used for IHC and Western Blot Analysis

Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

Techniques: Western Blot

Journal: The American Journal of Pathology

Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

doi: 10.1016/j.ajpath.2019.10.017

Figure Lengend Snippet: SFK substrates CUB-domain containing protein 1 (CDCP1) and protein kinase C δ (PKCδ) are phosphorylated in SFK_pY416 positive triple-negative breast cancer (TNBC). A: Representative patient sample with diffuse immunohistochemical staining of tumor cells for SFK_pY416, CDCP1_pY743, and PKCä_pY311. Staining for each marker was detected primarily on the cell membrane. Negative cells are predominantly stromal lymphocytes. B: Representative patient sample with a focal/low (<20%) pattern of staining for SFK_pY416, CDCP1_pY743, and PKCä_pY311 in tumor cells. C: Representative patient sample with negative staining. Insets: Areas of interest at higher magnification to show detail. D: Summary of staining results in panel of 56 TNBC samples. Main images were acquired using a 10× objective, and insets were acquired using a 40× objective. Scale bars = 100 μm (A–C). FOXA, forkhead box protein.

Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

Techniques: Immunohistochemical staining, Staining, Marker, Negative Staining

Journal: The American Journal of Pathology

Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

doi: 10.1016/j.ajpath.2019.10.017

Figure Lengend Snippet: Clinical and Pathologic Features of TNBC Patient Samples in Relation to High (> 20%) SFK_pY416 Expression

Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

Techniques:

Journal: The American Journal of Pathology

Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

doi: 10.1016/j.ajpath.2019.10.017

Figure Lengend Snippet: SFK_pY416 and SFK_pY527 are co-expressed in triple-negative breast cancer (TNBC). A: Western blot analysis confirms the co-expression of phosphorylated SFK/CUB-domain containing protein 1 (CDCP1) proteins, as determined by immunohistochemistry (IHC), and shows the co-expression of SFK_pY416 and SFK_pY527. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. forkhead box protein 1 (FOXA1), vimentin, and SFK_pY416 expression levels in tumor cells, as determined by IHC, are summarized on top. Probing with SFK_pY416 antibody detects a 60-kDa band in all patient samples that were SFK_pY416+ by IHC (lanes 4 and 8 to 13) and does not detect the band in patient samples that were SFK_pY416− by IHC. Cleaved CDCP1_pY734, CDCP1_pY743, and SFK_pY527 are co-expressed with SFK_pY416. Whole cell lysate from the TNBC cell line MDA-MB-231 was used as a positive control (lane 1). TNBC-2 (lane 4) has granular cytoplasmic staining for SFK_pY416. All other SFK_pY416+ tumors had membrane staining. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. B: Analysis of TNBC proteome in silico showing top eight proteins (protein names displayed next to data point) whose expression is correlated with SFK_pY416, with SFK_pY527 exhibiting the strongest correlation. TNBC tumor samples were isolated from The Cancer Proteome Atlas (TCPA) breast cancer (BRCA) data set by negativity for estrogen receptor, progesterone receptor, and human epidermal growth factor 2 receptor and analyzed for correlation of protein markers. Correlations were derived by Pearson product moment correlation test. cCDCP1, cleaved CDCP1; EGFR, epidermal growth factor receptor; FASN, fatty acid synthase; flCDCP1, full-length CDCP1; MEK1, dual specificity mitogen-activated protein kinase kinase 1; NFKBp65, nuclear factor NF-kappa-B p65 subunit; PEA15, astrocytic phosphoprotein PEA-15; RAB25, Ras-related protein rab-25; SFK, Src family kinase; SHP2, Src homology region 2–containing protein tyrosine phosphatase; STAT3, signal transducer and activator of transcription 3.

Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

Techniques: Western Blot, Expressing, Immunohistochemistry, Positive Control, Staining, Fluorescence, In Silico, Isolation, Derivative Assay

Journal: The American Journal of Pathology

Article Title: Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

doi: 10.1016/j.ajpath.2019.10.017

Figure Lengend Snippet: Western blot analysis of SFK_pY416− triple-negative breast cancer (TNBC) reveals that forkhead box protein 1 (FOXA1)+ samples tend to be SFK_pY527+ and FOXA1− samples tend to be SFK_pY527−. Paraffin sections of TNBC were extracted and analyzed by standard Western blot analysis technique. FOXA1, vimentin, and SFK_pY416 expression levels in tumor cells, as determined by immunohistochemistry (IHC), are summarized on top. A: All FOXA1+/SFK_pY416− samples tested were SFK_pY527+. The presence of SFK_pY416 in lanes 2, 4, and 6 is due to the presence of SFK_pY416+ lymphocytes in these tumor samples (shown in Supplemental Figure S1). B: Five of six FOXA1−/SFK_pY416− samples tested were SFK_pY527− (lanes 2, 3, and 5 to 7). Three FOXA1−/SFK_pY416+ tumor samples were run as controls. All samples contain phosphorylated AMP-activated protein kinase alpha (AMPKα) as a uniformly phosphorylated control for phosphoprotein extraction. Equal amounts of protein were loaded to each lane, as quantitated by tryptophan fluorescence spectrometry. β-Actin was used as a loading control. cCDCP1, cleaved CDCP1; CDCP1, CUB-domain containing protein 1; flCDCP1, full-length CDCP1; PKC, protein kinase C.

Article Snippet: CDCP1_pY734 , Cell Signaling Technology/9050 , 1:1000 , NA , NA.

Techniques: Western Blot, Expressing, Immunohistochemistry, Fluorescence