Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: AP2α targeting by miR-221&222. ( a ) WB analysis of AP2α with relative densitometric evaluation and miR-221 and -222 levels in normal melanocytes (NHEM) and melanoma cell lines. ( b ) Nucleotide pairing between AP2α 3′UTR and miR-221 and -222 is shown by bars. The seed sequence is indicated in bold, while lower case letters with asterisks represent the mutated nucleotides. ( c ) Luciferase reporter assay performed in 293FT (left) and Me1007 (right) cell lines by cotransfecting miR-221 or -222 in the presence of the AP2α 3′UTR. As controls, mutated 3′UTR sequences and a non-targeting oligomer were also included. ( d ) qRT–PCR (left) and representative WB analysis (right) of AP2α in Me1007 (top) and Me1402/R (bottom) control Tween vector vs miR-221- or 222-transduced cells. ( e ) Representative WB analysis of AP2α in anti-miR-221&222-transfected Me1402/R and A375M melanoma cells. GAPDH and actin are the internal controls. Non-targeting oligomers were also included as negative controls. Columns, mean±s.d. of at least three independent experiments. * P <0.05, ** P <0.01. LUC, luciferase; Mut, mutated; non targ, non-targeting; WB, western blot.

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Sequencing, Luciferase, Reporter Assay, Quantitative RT-PCR, Control, Plasmid Preparation, Transfection, Western Blot

Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: EGFL7/miR-126&126* promoter regulation. ( a ) Schematic illustration of the genomic region representing the promoter 1 of EGFL7. BS1-2, BS3, BS4 and BS5 indicate the AP2α BSs. ( b ) Promoter luciferase assays performed in the 293FT and Me1402/R cells transfected with AP2α. As control, the empty vector Tween was included. ( c ) Promoter luciferase assays performed in the 293FT and Me1402/R cells transfected with either Dsi-AP2α or Dsi-scr negative control. ( d ) As control of the APα BSs specificity, point mutations were inserted in the core-binding sequences and promoter luciferase assay performed. ( e ) Chromatin immunoprecipitation assay performed in Me1402/R cells with anti-AP2α antibodies and analyzed by semiquantitative PCR. Columns, mean±s.d. of at least three independent experiments. * P <0.05, ** P <0.01, *** P <0.001. Mut, mutated.

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Luciferase, Transfection, Control, Plasmid Preparation, Negative Control, Binding Assay, Chromatin Immunoprecipitation

Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: AP2α-dependent regulation of miR-126&126* and miR-221&222. qRT–PCR evaluations of ( a ) miR-126&126* (left), and miR-221&-222 (middle) in AP2α-overexpressing Me1402/R cell line and miR-126&126* after AP2α silencing (right). ( b ) miR-126&126* as a consequence of AP2α enforced expression or silencing in Me1007. ( c ) miR-126&126* in AP2α-transduced A375M cells. Samples were normalized on RNU6B levels. Columns, mean±s.d. of at least three independent experiments. ** P <0.01, *** P <0.001. ( d ) In situ hybridization of miR-221, miR-222 and miR-126 and immunohistochemistry of AP2α. Bar, 50 μm. Representative sections from one primary and one metastatic melanoma specimen are shown. Scrambled and RNU6B correspond to negative and positive controls, respectively. * P <0.05, ** P <0.01.

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Quantitative RT-PCR, Expressing, In Situ Hybridization, Immunohistochemistry

Journal: Oncogene

Article Title: AP2α controls the dynamic balance between miR-126&126* and miR-221&222 during melanoma progression

doi: 10.1038/onc.2015.357

Figure Lengend Snippet: AP2α expression is directly related with EGFL7 and miR-126&126* and inversely with miR-221&222. ( a ) Evaluation of EGFL7 in AP2α-transduced Me1402/R and A375M cells at mRNA (left) and protein (right) levels (** P <0.01). ( b ) Schematic depiction of miR-126&126* and miR-221&222 coregulatory pathways. The AP2α dependent activation of miR-126&126* and the consequential ADAM9 and MMP7-targeted downregulation prevent the pro-HB-EGF shedding in normal melanocytes (left). In melanoma the low levels of miR-126&126* unblock ADAM9 and MMP7 resulting in pro-HB-EGF shedding. This proteolytic cleavage originates the intracellular HB-EGF-C fragment that, entering the nucleus, binds and delocalizes the PLZF transcription factor, thus preventing its repressive function on miR-221&222 transcription. High amounts of miR-221&222 downregulate AP2α and consequently miR-126&126*, thereby contributing to close the circuitry maintaining advanced melanoma traits (right).

Article Snippet: The polyclonal rabbit anti -AP2α (Santa Cruz) was used as primary antibody at a dilution of 1:200 and the goat anti-rabbit IgG-HRP (656120; Life Technologies) at 1:3000 dilution as secondary one.

Techniques: Expressing, Activation Assay

Journal: Human Molecular Genetics

Article Title: Ap2s1 mutation causes hypercalcaemia in mice and impairs interaction between calcium-sensing receptor and adaptor protein-2

doi: 10.1093/hmg/ddab076

Figure Lengend Snippet: Co-immunoprecipitation analysis of the interaction between AP2σ2 and other subunits of the AP2 complex in HEK293 cells. ( A ) Immunoprecipitation using an anti-HA antibody and HEK293 cells stably expressing either: FLAG-tagged CaSR and HA-tagged WT AP2σ2 (WT); FLAG-tagged CaSR and HA-tagged mutant (m) Leu15 AP2σ2 (L15 (m)); or FLAG-tagged CaSR alone (-ve). The amount of protein in the total lysates and precipitated immune complexes (IP:HA-AP2σ2) was analyzed by western blotting using anti-HA, anti-AP2α, AP2β2, AP2μ2 and anti-tubulin antibodies. ( B – E ) Densitometry of the western blotting to quantify; (B) AP2σ2; (C) AP2α; (D) AP2β2 and (E) AP2μ2 in the immunoprecipitate of WT and L15 (m) cells, normalized to the amount of AP2σ2 in the total lysate (pre-normalized to tubulin). Mean ± SEM values are indicated, and data were analyzed using the Mann–Whitney U test. NS, non-significant; * P < 0.05.

Article Snippet: Proteins were transferred to polyvinylidene difluoride membrane and probed with primary antibodies (CASR ADD, Abcam), AP2σ2 (ab92380, Abcam), FLAG (ab49763, Abcam), HA (CST3724, Cell Signalling Technologies), AP2α (BD610501, BD Biosciences), AP2β2 (BD610381, BD Biosciences), AP2μ2 (ab137727, Abcam), tubulin (ab15246, Abcam) and calnexin (AB2301, Merck), and HRP-conjugated secondary antibodies (715-035-150 and 711-035-152, Jackson ImmunoResearch), prior to visualization using Pierce ECL Western blotting substrate.

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Mutagenesis, Western Blot, MANN-WHITNEY