Research Paper Volume 12, Issue 20 pp 20268—20284

TFG-maintaining stability of overlooked FANCD2 confers early DNA-damage response

Figure 2. TFG interacts with FANCD2-V2, but not FANCD2-V1. (A) TFG interacts with the endogenous FANCD2-V2, but not FANCD2-V1. Reverse immunoprecipitation (IP) and western blotting (WB) of endogenous FANCD2-V2, FANCD2-V1 and TFG Rabbit IgG were performed using lysates prepared from U2OS cells. TFG signaling can be clearly detected in the pulldown by antibodies recognizing FANCD2-V2 but not FANCD2-V1. (B) TFG is co-peaked with FANCD2-V2 but not with FANCD2-V1. Gel filtration was performed using the total cell lysates prepared from U2OS cells. Western blotting of FANCD2-V1, -V2 or TFG showed that TFG and FANCD2-V2 were peaked at the same fraction, but FANCD2-V1’s peak was a fraction late. (C) Fluorescent co-localization of TFG with FANCD2-V2, not with FANCD2-V1. Immunofluorescent study was performed on normally growing U2OS cells. The fixed cells were primarily blocked with antibodies targeting FANCD2-V2, FANCD2-V1, or TFG. Subsequently, anti-Rabbit-Alexa 488 and anti-mouse-Alexa 568 were applied (DAPI was used for nuclear staining. Green fluorescence of FANCD2-V2, but not FANCD2-V1, can be merged with the red florescence of TFG (the orange color). The association of TFG with FANCD2-V2 was also supported by Pearson’s or Mander’s colocalization coefficient with an R nearly 1 (0.96 or 0.98). (D) The association of FANCD2-V2 with TFG is attributed to its unique carboxyl terminal. HEK293T cells were transfected with empty vectors, GFP-wtFANCD2-V2, GFP-mtFANCD2-V2Δaa1437-1442, GFP-mtFANCD2 K561R or GFP-wtFANCD2-V1. Antibodies targeting GFP or endogenous TFG were used for reverse IP and WB. TFG can pull down GFP-wtFANCD2-V2, point mutant of FANCD2-V2K561R, but not the carboxyl terminal deletion mutant of FANCD2-V2, either GFP-wtFANCD2-V1 (IgG negative control was performed simultaneously-Supplementary Figure 2D).