Journal: Cell Death & Disease

Article Title: Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

doi: 10.1038/cddis.2017.406

Figure Lengend Snippet: Anlysis of selected mRNA and protein expression alterations in the BRAFi-resistant cell lines compared with A375 parental cells

Article Snippet: Cell membrane was permeabilized with 0.1% triton X100 diluted in PBS and for minimizing the risk of unspecific binding, cells were treated with blocking buffer containing 5% horse serum diluted in PBS for 1 h. Then cells on coverslip were incubated overnight with primary antibody against pAb anti-CD13 (ab7417 Abnova diluted 1:100 in blocking buffer, Taipei, Taiwan) at 4 °C overnight.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells

doi: 10.1038/cddis.2017.406

Figure Lengend Snippet: Potential BRAFi resistance candidates. Blocking of CD13/ANPEP leads to dephosphorylation of EPHA2 on S897 and increased apoptosis. ( a ) Protein expression levels of the potential resistance candidate protein CD13/ANPEP analyzed by immunoblotting in presence of 1 μ M of vemurafenib or DMSO as control treatment for 24 h in parental A375 and three BRAFi-resistant sublines. β -actin was used as control. ( b ) Immunofluoroscence showing CD13 expression in A375, A375VR4 and SKMEL24 cells. CD13 is stained with Alexa Fluor 594 (red) and nucleus is counterstained with DAPI (blue). ( c ) EPHA2, AKT1 and RSK phosphorylation was decreased after 48 h treatment with anti-CD13 blocking antibody. ( d ) Induction of apoptosis after 48 h treatment with anti-CD13 blocking antibody in A375, A375VR4 and SKMEL24 cells. Apoptosis was measured by flow cytometry based annexin V-PI staining. (Error bars represent S.D. of the mean of two independent experiments each in three replicates)

Article Snippet: Cell membrane was permeabilized with 0.1% triton X100 diluted in PBS and for minimizing the risk of unspecific binding, cells were treated with blocking buffer containing 5% horse serum diluted in PBS for 1 h. Then cells on coverslip were incubated overnight with primary antibody against pAb anti-CD13 (ab7417 Abnova diluted 1:100 in blocking buffer, Taipei, Taiwan) at 4 °C overnight.

Techniques: Blocking Assay, De-Phosphorylation Assay, Expressing, Western Blot, Control, Staining, Phospho-proteomics, Flow Cytometry