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. 2014 Sep 5;33(21):2521–2533. doi: 10.15252/embj.201488663

Figure 5. Depletion of FANCJ results in persistent stalling at G-quadruplex structures.

Figure 5

  1. Mock-depleted, FANCJ-depleted, and FANCJ-depleted HSS supplemented with recombinant xlFANCJ were analyzed by Western blotting using xlFANCJ antibody. A dilution series of undepleted extract was loaded on the same blot to determine the degree of depletion. A relative volume of 100 corresponds to 0.7 μl of HSS. A non-specific band cross-reacting with FANCJ antibody is used as a loading control (‘Loading’).
  2. Mock-depleted, FANCJ-depleted, and FANCJ-depleted HSS supplemented with recombinant xlFANCJ were used to replicate the G4G3N plasmid template starting from primer A. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography. Products stalled at the G4 sequence (‘stalled’), linear molecules resulting from denatured nicked products (‘linear’), and closed supercoiled products (‘supercoiled’) are indicated.
  3. G4G3N was replicated in FANCJ-depleted HSS starting from primer A. After 60 min, xlFANCJ or buffer was added followed by an additional 30-min incubation. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography.
  4. Mock-depleted and FANCJ-depleted HSS were used to replicate G4G3N starting from primer A in the absence or presence of a low concentration (0.75 μM) of Phen-DC3. Replication products were extracted, separated on 6% urea-PAGE gels, and visualized by autoradiography.
  5. The G4 stalled and bypassed products of the 90-min time point in (D) were quantified using ImageQuant software, and the percentage of stalling versus bypassed products was plotted for the various conditions. Black bars represent percentage of stalling in the mock-depleted samples, the sum of black and dashed bars represent the percentage of stalling in the FANCJ-depleted samples. Therefore, dashed bars represent percentage of stalling as a result of FANCJ depletion only.