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. 2013 Jan 28;110(7):2523–2527. doi: 10.1073/pnas.1222494110

Fig. 3.

Fig. 3.

Interaction of Tim-Tipin with the CMG complex and its influence on the DNA helicase activity of the CMG complex. (A) Equivalent amounts of Tim-Tipin and CMG complexes (0.5 pmol) were combined and immunoprecipitated with antibodies against Mcm2 or Tim. Precipitated proteins were analyzed by Western blot analysis. Lanes 1 and 4 represent input materials (10%) for immunoprecipitation. Asterisks indicate bands of Ig heavy chains. (B) (Upper) DNA helicase activity of CMG complex (33 fmol) was measured after incubation in the absence or presence of increasing levels of Tim-Tipin, Tim, or Tipin (16.5, 33, 66, and 99 fmol) at 4 °C for 1 h. Lane 1, boiled substrate; lane 2, no protein added; lane 3, CMG alone; lanes 4–15, CMG with Tim-Tipin, Tim, or Tipin proteins. (Lower) Quantitation of the duplex unwinding activity shown in the upper panel. Relative DNA unwinding activity was calculated as percent activity observed compared with the activity detected with CMG complex alone (shown in B, lane 3).