Journal: Oncogene

Article Title: Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis

doi: 10.1038/onc.2009.328

Figure Lengend Snippet: (A) The expression of various myosin VI binding proteins was compared between normal prostate cells (PNT1A) and LNCaP cells by Western blotting. Calreticulin was used as a loading control. The expression of Dab2 is completely lost in LNCaP cells, whereas the expression of optineurin is downregulated. (B) Co-immunoprecipitation of myosin VI with LMTK2 or GIPC from LNCaP cells. Myosin VI was immunoprecipitated using polyclonal antibodies against the myosin VI tail. A control immunoprecipitation was performed with polyclonal antibodies to the androgen receptor (AR). The lysate lane contains about 5 % (GIPC and LMTK2) or 1 % (myosin VI) of the input used for each immunoprecipitation. The immunoprecipitated protein complexes were blotted with antibodies against LMTK2, GIPC or myosin VI. A quarter of the immunoprecipitated myosin VI is shown on the blot. (C) Expression of Dab2 in LNCaP cells rescues recruitment of myosin VI to clathrin coated structures at the plasma membrane. Untransfected LNCaP cells (a) (−Dab2) or LNCaP cells transfected with full-length Dab2 (d) (+Dab2) were processed for immunofluorescence microscopy and labelled with anti myosin VI (a and d) and anti clathrin (b and e) antibodies. In cells transfected with the Dab2 construct a dramatic colocalisation between endogenous myosin VI (d) and clathrin (e) can be observed in the merged image (f). (D) Endogenous myosin VI coimmunoprecipitates with GFP-Dab2 from LNCaP cells. GFP-Dab2 was transiently expressed in LNCaP cells and immunoprecipitated using polyclonal GFP antibodies. The immunoprecipitated complexes were blotted with antibodies against myosin VI or Dab2.

Article Snippet: Antibodies used: GFP (Molecular Probes), AP2 (AP6, ABR), Rab5 (BD Transduction Laboratories), TfR (Zymed), TGN46 (Serotec), Dab2, androgen receptor, calregulin, calnexin, EGFR (all from Santa Cruz), anti-GIPC (Proteus Biosciences), clathrin (Abcam), PSA (Sigma), myosin VI tail , T6BP and NDP52 , optineurin ( ) and LMTK2 ( ).

Techniques: Expressing, Binding Assay, Western Blot, Control, Immunoprecipitation, Clinical Proteomics, Membrane, Transfection, Immunofluorescence, Microscopy, Construct

Journal: Oncogene

Article Title: Overexpression of myosin VI in prostate cancer cells enhances PSA and VEGF secretion, but has no effect on endocytosis

doi: 10.1038/onc.2009.328

Figure Lengend Snippet: (A) Immuno-EM showing the localisation of PSA in the perinuclear region in LNCaP cells. Cryosections of LNCaP cells were either labelled with antibodies against endogenous PSA (a) (15 nm gold), with antibodies against PSA (10 nm gold) and the TfR (15 nm gold) (b) or with antibodies to PSA (10 nm gold) and to myosin VI (15 nm gold) (c). PSA colocalises with TfR (b) and myosin VI (c) on tubular/vesicular membranes in the perinuclear region close to the Golgi complex. Bar: 340 nm. (B) To reduce expression of myosin VI in LNCaP cells, the cells were transfected twice with siRNA targeting myosin VI or with non-specific control siRNA (mock). Two days after the second transfection myosin VI depletion was assessed by immunoblotting with antibodies to myosin VI and to calregulin as a loading control. (C) The medium of mock transfected and myosin VI siRNA knock down cells was collected and the amount of secreted PSA was measured using a commercially available ELISA kit. (D) To assess the role of different myosin VI isoforms for PSA secretion, LNCaP cells were transfected with control pEGFP vector or with the different dominant negative tail isoforms of myosin VI: GFP-tail NI, GFP-tail LI-abc, GFP-tail LI-ab or GFP-tail LI-bc. Transfected cells expressing GFP-tagged tails were isolated by FACS sorting and grown for 48 h before the PSA concentration in the medium was determined by ELISA.

Article Snippet: Antibodies used: GFP (Molecular Probes), AP2 (AP6, ABR), Rab5 (BD Transduction Laboratories), TfR (Zymed), TGN46 (Serotec), Dab2, androgen receptor, calregulin, calnexin, EGFR (all from Santa Cruz), anti-GIPC (Proteus Biosciences), clathrin (Abcam), PSA (Sigma), myosin VI tail , T6BP and NDP52 , optineurin ( ) and LMTK2 ( ).

Techniques: Expressing, Transfection, Control, Western Blot, Knockdown, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Dominant Negative Mutation, Isolation, Concentration Assay