Figure 2.

RTEL1 Interacts with TRF2 through Cysteine-Rich C4C4 Domain

(A) Immunoblotting of a peptide array for the C-terminal 1147–1369 amino acids of human RTEL1. The arrays were incubated either with GST or with human TRF2 fused with GST, and bound proteins were examined by immunoblotting against GST.

(B) Whole-cell extracts from HEK293 cells were incubated in the absence (−EDTA) or presence (+EDTA) of 50 mM EDTA either with GST or the C4C4 domain (corresponding to human RTEL1_1234–1292 region) fused to GST. The bound complexes were immobilized to glutathione-linked Sepharose beads followed by western blotting analysis against TRF2 and GST.

(C) Schematic representation of mammalian RTEL1 protein. Full-length RTEL1 is segmented into functional domains shown as colored boxes. Asterisks indicate locations of mutations introduced into human or mouse C4C4 domain.

(D) HEK293 cells were stably transduced with lentiviruses carrying an empty vector (ctrl) or full-length versions of the wild-type Flag-RTEL1 (WT), single-mutant Flag-RTEL1^R1264H (R/H), or double-mutant Flag-RTEL1^{C1279A/C1282A} (C/A) as indicated. Whole-cell extracts were immunoprecipitated with anti-TRF2 antibody, and western blotting was carried out to detect the interaction between TRF2 and Flag-tagged RTEL1.

(E) Cell extracts as indicated in (D) were incubated with GST control or human TRF2 fused to GST. The protein complexes were immobilized to glutathione-linked Sepharose beads followed by western blotting analysis against Flag and GST.

(F) Whole-cell extracts of MSK-41 fibroblasts and BJ-hTERT control fibroblasts were immunoprecipitated with anti-RTEL1 antibody. Immunocomplexes were analyzed with antibodies as indicated.

(G) The well-characterized interaction between TRF2 and Rap1 was included as a specificity control for the PLA assay. The graphs depict quantification of the interaction spots per cell as determined by PLA using antibodies as indicated.

(H) Quantification of the interaction between TRF2 and RTEL1 as determined by in situ PLA assay in MSK-41, RPE-1, and BJ-hTERT cells with indicated antibodies. Dashed lines indicate nucleus outlines (as determined by DAPI in blue). Data in (G) and (H) are representative of two independent experiments as mean ± SD (^∗∗∗∗p < 0.0001, one-way ANOVA). See also Figure S3.