Phallo din tritc

BL3 and TBL3 cells were plated on Fibronectin coated slides (Sigma; Ref: F1141). NIH/3T3 cells and Mouse Immortalized Fibroblasts cells-transfected by indicated constructs were plated on slides. All cells were then fixed in PBS 3.7% Formaldehyde for 15min at room temperature. Slides with bovines cells or NIH/3T3 cells were rinsed in PBS and permeabilized with PBS 0.2% Triton X-100 for 5min and then blocked for 30min with PBS 1% SVF and 1% BSA to prevent non-specific staining. These slides were incubated with rabbit anti-TaPin1 (1/250) and/or mouse anti-HA (1/1000, Roche, Ref: 11583816001) in PBS 1% SVF and 1% BSA at room temperature for 40min. After washing in PBS 0.2% Tween, the slides were incubated with Texas Red dye-conjugated AffinyPure Donkey anti-rabbit IgG and/or Cy2 AffinyPure Donkey anti-mouse IgG (1/5000, Jackson Immunology, Ref: 711-075-152 or Ref: 715-225-150) for 30min. Slides with Murine Immortalized Fibroblasts cells were incubated for 15min with Phalloïdin-TRITC (Life Technologies, Ref: R415). All slides were subsequently washed in PBS 0.2% Tween, mounted on slides and covered with ProLong Gold Antifade Reagent with DAPI (Invitrogen, Ref: P-36931). Images of immunofluorescence staining (Mouse Immortalized Fibroblasts cells) were photographed with a fluorescent microscope (Leica Inverted 6000). Staining was repeated for three independent biological replicates.