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. 2020 Aug 1;34(15-16):1065–1074. doi: 10.1101/gad.330050.119

Figure 1.

Figure 1.

RTEL1 associates with Poldip3. (A) Schematic flow chart of the Flag affinity purification protocol of Flag-RTEL1. Flag-CHFR or Flag-RTEL1 or expressing HeLa cells were harvested and whole cell lysates were treated with a cocktail of DNases and RNases. Flag-RTEL1 fusion proteins were Flag immunopurified, eluted with Flag peptides, and immunocomplexes resolved by PAGE. (B) Silver staining of resolved proteins from the Flag-RTEL1 immunoprecipitation. Control immunoprecipitation was performed with a Flag-CHFR fusion protein. CHFR was used as control since it, like RTEL1, is a RING finger-containing protein. Flag-CHFR, Flag-RTEL1 and Poldip3 are indicated with arrows. (C) GFP-RTEL1 binds HA-Poldip3. HEK293T cells were cotransfected with HA-Poldip3 and either GFP or GFP-RTEL1 and incubated for 24 h prior to harvest. Extracts were subjected to GFP immunoprecipitation followed by immunoblotting with HA and GFP antibodies. (D) Endogenous RTEL1 binds endogenous Poldip3. U2OS cells were harvested in EBC buffer and cell lysates were treated as in A. Precleared lysates were subjected to immunoprecipitation with either IgG or RTEL1 antibody. RTEL1 immunocomplexes were released from the RTEL1-conjugated resin by treatment with glycine-HCl buffer.