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. 2015 Apr 10;34(10):1385–1398. doi: 10.15252/embj.201490376

Figure 2.

Figure 2

UBL5 is required for the integrity of the FA pathway
  1. Extracts of HeLa cells transfected with non-targeting control (CTRL) or UBL5 siRNAs for 48 h were analyzed by immunoblotting with the indicated antibodies. * non-specific band.
  2. HeLa cells stably expressing a doxycycline-inducible siRNA-resistant form (siR) of Strep-HA-UBL5 were transfected with non-targeting control (CTRL) or UBL5 siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with FANCI, FANCD2 and β-tubulin antibodies.
  3. HeLa cells transfected with non-targeting or UBL5 siRNAs were transfected with Strep-HA-UBL5 or siRNA-resistant form (siR) of Strep-HA-UBL5. Cell extracts were analyzed by immunoblotting with HA and β-actin antibodies.
  4. HeLa cells transfected with non-targeting control (CTRL) or UBL5 siRNAs and then treated or not with MMC for 15 h were lysed and processed for immunoblotting with FANCI, FANCD2, UBL5 and β-tubulin antibodies.
  5. U2OS cells transfected with non-targeting control (CTRL) or UBL5 siRNAs were subjected to laser microirradiation in the presence of trioxsalen, fixed 2 h later and co-immunostained with FANCI, γ-H2AX and cyclin A antibodies. Representative images are shown. Scale bar, 10 μm.
  6. Clonogenic survival of U2OS cells transfected with indicated siRNAs and exposed to various doses of MMC. Results from at least three independent experiments (mean ± SEM) are shown.
  7. Clonogenic survival of U2OS/Strep-HA-UBL5(siR) cells treated or not with doxycycline (DOX) to induce expression of the transgenes, transfected with the indicated siRNAs and then exposed to various doses of MMC. Results from three independent experiments (mean ± SEM) are shown.