DEV02687 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Csul physically interacts with Tud. (A) Map of the Tud protein containing eight Tudor (1-8) and two Tudor-like domains (1’,2’) depicted as purple boxes. Fragments of Tud (Golumbeski et al., 1991) used in the pull-down assay are indicated below the map. N- and C-terminal amino acid residues of each fragment are indicated. (B) Immunoblot of S-Tag-Tud fragments detected using alkaline phosphatase-conjugated S proteins. (This blot is identical to that shown in fig. 6B of Anne and Mechler (Anne and Mechler, 2005) because the same reagents were used for the binding assays). (C) After incubation with GST or GST-Csul, the bound Tud fragments were detected by immunoblotting using alkaline phosphatase-conjugated S proteins. The Tud-JOZ, Tud-9A1-N and Tud-9A1-C fragments display a strong binding to GST-Csul, whereas Tud-3ZS+L-N binds only weakly to it and Tud-3ZS+L-C does not bind. GST alone exhibits no binding to Tud-JOZ and Tud-9A1. (Lower panel) Input GST and GST-Csul proteins separated by SDS-PAGE and stained with Coomassie Blue.

• Supplemental Figure 2 -

  Fig. S2. Methylated SmB binds to Tud in vitro. (A) In the reticulocyte lysate system, SmB is symmetrically methylated by an endogenous methyltransferase. ³⁵S-methionine-labelled SmB proteins were synthesized in presence or absence of the protein methyltransferase inhibitor, S-adenosyl-homocysteine (SAH), separated by SDS-PAGE, blotted and probed with α-SYM10 antibodies. The position of SmB is indicated with an arrow. Synthesis of radioactively labelled SmB proteins in the presence of SAH is shown in the lower panel. (B) Expression of GST-Tud in bacteria. Fragments of Tud (Golumbeski et al., 1991) including JOZ, 9A1 and 3ZS+L, encompassing residues 3-273, 198-1199 and 1198-2515, respectively, were separated by SDS-PAGE and stained with Coomassie blue. (C) Binding of sDMA-SmB to Tud. Following incubation with GST-Tud, fragments of the bound ³⁵S-SmB proteins, synthesized as described above, were separated by SDS-PAGE and detected by fluorography. Only sDMA-SmB proteins bind to all three Tud fragments, presumably through the Tudor domains.