Journal: Developmental cell
Article Title: The Nf2 Tumor Suppressor, Merlin, Regulates Epidermal Development Through the Establishment of a Junctional Polarity Complex
Figure Lengend Snippet: The interaction of Merlin with both β-catenin and Par3 is required for the establishment of functional junctions (A) IVTT-produced Merlin or Merlin 18-595 was mixed with either GST-β-catenin (top) or Myc-Par3 (middle). Complexes were isolated using either GST beads (top panel) or an anti-Myc antibody (middle panel) and immunoblotted with anti-GST, -Myc, or -Nf2 antibodies. Merlin 18-595 can readily bind Myc-Par3 but not GST-β-catenin in vitro . 10% of the input is shown in the bottom panel. (B) PAM212 keratinocytes were transfected with Myc-Par3, Myc-Par3 511-1266 or Myc-Par3 1-373 and cultured in calcium-containing media for the indicated times. Immunoprecipitation of Par3 followed by immunoblotting with Myc-, AJ- or TJ-specific antibodies revealed that full-length Myc-Par3 and Myc-Par3 511-1266 can associate with Merlin and with AJ and TJ proteins. In contrast, Myc-Par3 1-373 does not associate with Merlin or with AJ and TJ proteins. Note that Myc-Par3 511-1266 and Myc-Par3 1-373 are produced from a Par3 splice variant that does not contain the aPKC-binding site. NRS, normal rabbit serum. (C) TER was measured across calcium-stimulated PAM212 keratinocyte monolayers that stably express Merlin, Merlin 18-595 , or Myc-Par3 1-373 . Note that both Merlin 18-595 and Myc-Par3 1-373 dominantly interfered with the establishment of TER. Values = mean +/− SD. (D–G) Primary wild-type (D and F) or K14-Cre;Nf2 lox/lox (E and G) keratinocytes were stimulated with calcium-containing media for 8 hours, and either processed for indirect immunofluorescence (Fix 1, D and E) or subject to a more stringent in situ extraction (Fix 2, F and G). Immunostaining revealed normal localization of Par3 (green) at cell-cell boundaries in K14-Cre;Nf2 lox/lox .
Article Snippet: For IP and western blot the following antibodies were used: NF2 (1:1000, A-19 and C-18, Santa Cruz), Keratin 14 (1:5000, AF64, Covance), Actin (1:1000, AC40, Sigma), β-tubulin (1:1000, SAP4G5, Sigma), Par3 (1:500, 07-330, Upstate), aPKC (1:500, C-20, Santa Cruz), HA (1:1000, clone 12CA5), Myc (1:1000, 9E10, Santa Cruz), ZO-1 (1:500, Z-R1, Zymed), E-cadherin (1:2000, #36, Pharmingen), β-catenin (1:2000, #14, Pharmingen), and α-catenin (1:1000, #5, Pharmingen).
Techniques: Functional Assay, Produced, Isolation, In Vitro, Transfection, Cell Culture, Immunoprecipitation, Variant Assay, Binding Assay, Stable Transfection, Immunofluorescence, In Situ, Immunostaining