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1) Product Images from "Tankyrase Polymerization Is Controlled by Its Sterile Alpha Motif and Poly(ADP-Ribose) Polymerase Domains"
Article Title: Tankyrase Polymerization Is Controlled by Its Sterile Alpha Motif and Poly(ADP-Ribose) Polymerase Domains
Journal: Molecular and Cellular Biology
Figure Legend Snippet: Dissociation of tankyrase complexes by tankyrase autoribosylation. (A) Solubility of purified full-length tankyrase 1 after incubation with NAD + . Samples were incubated with (+) or without (−) 1 mM NAD + for 10 or 30 min at 25°C and centrifuged to separate soluble (S) from insoluble (P) material. Tankyrase was detected by Western blotting with antibody to tankyrase (α-TNK) or poly(ADP-ribose) (α-PAR). Arrow, position of unmodified tankyrase. (B) Solubility of overexpressed tankyrase 1 in LMH cell extracts. Cells expressing FL-tankyrase1 (TNK) or catalytically inactive FL-tankyrase1 (TNK-PARP † ) were lysed at 25°C in the presence (+) or absence (−) of 1 mM NAD + and/or 10 mM 3AB and centrifuged. Tankyrase was detected in the supernatant (S) or pellet (P) by Western blotting with tankyrase antibody.
Techniques Used: Solubility, Purification, Incubation, Western Blot, Expressing
Figure Legend Snippet: Effects of tankyrase overexpression on the secretory pathway. (A and B) Colocalization of tankyrase vesicles with FTCD. Control LMH cells (A) or LMH cells expressing Myc-tagged FL-tankyrase1 (B) were fixed and incubated with tankyrase antibody (A1), Myc antibody (B1), or FTCD antibody (A2 and B2). Panels A3 and B3 show an overlay of the tankyrase (green) and FTCD (red) signals. Cells were counterstained with DAPI. wt, wild type. (C and D) Effect of Myc-FL-tankyrase1 on secretion of marker proteins. LMH cells were transfected with GPI-GFP or VSVG3-GFP and analyzed by fluorescence microscopy after 30 h at 37°C (GPI-GFP) or 32°C (the permissive temperature for temperature-sensitive VSVG3-GFP). (C) Localization of GPI-GFP and VSVG3-GFP in wild-type cells. (D and E) Localization of GPI-GFP and VSVG3-GFP after tankyrase overexpression. Cells were transfected with Myc-tagged FL-tankyrase1 and GPI-GFP or VSVG3-GFP. (D1 and E1) Exogenous tankyrase detected with Myc antibody; (D2 and E2) GPI-GFP and VSVG3-GFP staining; (D3 and E3) overlay of panels 1 and 2. Red, tankyrase; green, GPI-GFP and VSVG3-GFP.
Techniques Used: Over Expression, Expressing, Incubation, Marker, Transfection, Fluorescence, Microscopy, Staining
Figure Legend Snippet: In vivo complex formation. (A) Solubility of overexpressed tankyrase 1 in LMH cell lysates. Cells expressing FL-tankyrase1 (TNK) or tankyrase1-ΔSAM (ΔSAM) were lysed at 4 or 25°C and centrifuged, and tankyrase was detected in the supernatant (S) or pellet (P) by Western blotting with tankyrase antibody. (B) Superose 6 fractionation of soluble tankyrase extracted from cells expressing FL-tankyrase1 (TNK) or tankyrase1-ΔSAM (ΔSAM) or from untransfected cells (endo). The total amount of protein loaded on the column was ∼250 μg from TNK- and ΔSAM-expressing cells and ∼650 μg from wild-type cells. The bands corresponding to the input material are shown in the lane to the left; the amounts loaded were 1% of the total for TNK, 2% for SAM, and 10% for wild-type extracts. The peak fractions containing marker proteins are shown at the top, and fraction numbers are shown below. Detection was by Western blotting with tankyrase antibody.
Techniques Used: In Vivo, Solubility, Expressing, Western Blot, Fractionation, Marker
Figure Legend Snippet: In vivo tankyrase localization. (A) LMH cells expressing Myc-tagged FL-tankyrase1 (Myc-Tankyrase), Myc-tagged tankyrase1-ΔSAM (Myc-ΔSAM), or Flag-tagged SAM domain (Flag-SAM) were fixed and incubated with antibody to the Myc or Flag tags. Nuclei were counterstained with DAPI. (B) Untransfected cells were stained with antibody to the tankyrase SAM domain (panel 1), tankyrase antibody that had been preincubated with purified SAM domain (panel 2), or secondary antibody (panel 3). (C) Cells coexpressing Myc-tagged FL-tankyrase1 and Flag-tagged SAM domain. Panel 1, staining with Myc antibody; panel 2, staining with Flag antibody; panel 3, combination of the Myc (green) and Flag (red) signals. (D) Examples of the different size of vesicle-like structures observed in FL-tankyrase1-expressing cells. Detection was with the Myc antibody.
Techniques Used: In Vivo, Expressing, Incubation, Staining, Purification