High Mobility of Flap Endonuclease 1 and DNA Polymerase Associated with Replication Foci in Mammalian S-Phase Nucleus

Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Colocalization of GFP-Fen1 and GFP-Pol with endogenous PCNA in S-phase cells. In (A) and (B) pre-extraction of transiently transfected cells was with CSK buffer containing 0.5% Triton X-100 (Kamiuchi et al., 2002), and in (C) pre-extraction of cells stably expressing GFP-Fen1 was with 100% methanol at -20oC (Madsen and Celis, 1985). PCNA was detected as described in Materials and Methods.
• Supplemental Figure 2 - Colocalization of GFP-CSA protein with phosphorylated RNA polymerase II in OS-7 cells pre-extracted with CSK buffer with 0.5% Triton X-100. RNA polymerase II was detected with monoclonal mouse antibodies clone H5 as described in Materials and Methods. Bar is 10 microns. Triangles mark completely colocalized domains.
• Supplemental Figure 3 - Inhibition of GFP-Fen1 mobility in transiently transfected V79 cells by treatment with 0.03% MMS. Vertical bars show SE obtained from 10 ? 20 cells.
• Supplemental Figure 4 - Induction of -H2AX foci in V79 cells by bleomycin. Treatment with bleomycin (60 g/ml) was for 2 hours, and then cells were fixed in 4% formaldehyde. Phosphorylated histone -H2AX was detected as described earlier (Tomilin et al., 2001) using secondary anti-rabbit IgG antibodies coupled to Alexa Fluor 568 (Molecular Probes) producing red signal, and DNA was visualized using SYBR Green (Molecular Probes). Bar is 20 microns.

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