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. 2008 Jul 25;283(30):20925–20936. doi: 10.1074/jbc.M802696200

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Copyright © 2008, The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 6. — hChlR1 interacts with the Ctf18-RFC complex and PCNA. A, in the three upper panels, 1 m g of lysates from 293 cells transiently expressing FLAG-Ctf18 (second panel from top) or FLAG-Dcc1 (third panel from top) alone (lane 2) or together with constitutively expressed ChlR1-HA (lane 4) were incubated with HA antibody as indicated at the top of the immunoblots; specific interactions were detected by Western blotting, using antibodies to hChlR1 (top panel), Ctf18 (second panel from top), or Dcc1 (third panel from top). In the bottom panel, lysates from 293 cells (lanes 2) or 293 cells constitutively expressing ChlR1-HA (lanes 4) were incubated with HA antibody, and specific interactions between ChlR1-HA and endogenous p37/RFC2 subunit were detected by Western blotting using p37/RFC2 antibody. B, reciprocal immunoprecipitations in 293 cells expressing both ChlR1-HA and FLAG-tagged Ctf18 performed with protein A beads in combination with antibodies specific for Ctf18 (lane 2) or with preimmune serum (lane 3), as indicated at the top of the immunoblot; specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and Ctf18 (bottom). C, immunoprecipitation using FLAG beads of lysates from 293 cells transiently expressing ChlR1-HA alone (lane 4) or together with FLAG-tagged Dcc1 (lane 3); specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and Dcc1 (bottom). D, hChlR1 does not interact with RFC1. Lysates from 293 cells constitutively expressing ChlR1-HA (lane 3) were incubated with HA antibody; no specific interaction was detected by Western blotting using antibodies to hChlR1 (top) and the RFC1 subunit (bottom). E, hChlR1 interacts with PCNA. Left, 1 mg of 293 cell lysates transiently expressing FLAG-ChlR1 (lane 2) or 293 cell lysates (lane 3), supplemented with recombinant HA-PCNA (5.6 pmol) were incubated with FLAG-M2 antibody beads; right, 1 mg of 293 cell lysates transiently expressing FLAG-ChlR1 in the presence (lane 5) or absence (lane 6) of recombinant HA-PCNA (5.6 pmol) were incubated with HA antibody beads; specific interactions were detected by Western blotting using antibodies to hChlR1 (top) and PCNA (bottom). In all immunoprecipitations, input represents 10% of the total amount of lysate/recombinant protein used for immunoprecipitation. IP, immunoprecipitation.