Fig 5.

Immunofluorescence analyses of LANA-1, ANG, and p53 interactions and mapping of ANG domain interacting with LANA-1 and p53. (A) Permeabilized TIVE-LTC cells were stained for ANG (green), LANA-1 (red), and p53 (blue). The red-bordered box image from a LANA-1-negative cell is merged, and the enlarged image of p53 and ANG is shown on the left. Red arrows point to p53 and ANG colocalization. (B) The white-bordered box image from a LANA-1-positive cell was merged, and enlarged images of double and triple colocalization of LANA-1, ANG, and p53 are shown. White arrowheads indicate the triple colocalization of LANA-1, ANG, and p53 (white spots), while the long white arrows point to LANA-1 and ANG colocalization (yellow spots). A Venn diagram indicating the effects of different color combinations is provided to interpret double and triple colocalization results. 1, LANA-1 and ANG; 2, LANA-1 and p53; 3, LANA-1, ANG and p53; 4, Ang and p53. (C) Schematic diagram showing full-length (FL) angiogenin indicating the amino acid (aa) residues with reported important functions, nuclear localization signal area, and N- and C-terminal deletion constructs. (D) 293T cells were transfected with FL ANG-GFP or four C-terminal GFP-tagged ANG deletion constructs, and their expression was checked by Western blotting for GFP. (E) 293T cells were transfected with full-length LANA-1 along with the empty vector, full-length ANG, or four ANG deletion constructs. The lysates were subjected to IP with rabbit anti-LANA-1 IgG antibodies, separated with a 10% gel, and subjected to Western blotting for GFP. LANA-1 expression levels were checked by Western blotting for LANA-1 in the samples. (F) 293T cells were transfected with the empty vector, FL ANG-GFP, or four of its deletion constructs, and the lysates were subjected to IP with rabbit anti-p53 IgG antibodies, separated with a 10% gel, and subjected to Western blotting for GFP. Input control for p53 and β-actin loading controls are shown.