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. 2022 Mar 15;82(10):1937–1952. doi: 10.1158/0008-5472.CAN-21-3038

Figure 4.

Figure 4. DHX37 interacts with PLRG1, a core component of the CDC5 L complex, in liver cancer cells. A, DHX37-associated proteins were analyzed with STRING. The network nodes represent potential interacting proteins obtained by Co-IP/LC/MS. The edges represent protein–protein interactions. B, Co-IP using control IgG and an anti-Flag (DHX37) or anti-HA (PLRG1) antibody was carried out using extracts prepared from the HEK293T cells transfected with the CMV-Flag-DHX37 and pCMV-HA-PLRG1 plasmids. The presence of DHX37 or PLRG1 in these immunoprecipitates was evaluated by immunoblot (WB) analysis. C, Co-IP using control IgG and an anti-DHX37 or anti-PLRG1 antibody was carried out using extracts prepared from Huh7 cells. The endogenous interaction of PLRG1 with DHX37 was evaluated by WB analysis with the corresponding antibodies. D, Huh7 and HepG2-C3A cells stably expressing DHX37 were fixed for immunofluorescence analysis. DHX37 was detected using an anti-Flag (DHX37) primary antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody, and PLRG1 was detected using an anti-PLRG1 primary antibody and an Alexa Fluor 594 goat anti-rabbit secondary antibody. Representative cells from the same field for each experimental group are shown. Scale bars: Huh7, 50 µmol/L (first column) or 25 µmol/L (other four columns). HepG2-C3A, 25 µmol/L (first column) or 10 µmol/L (other four columns). E, Schematic representation of Flag-tagged full-length DHX37 (FL) along with its various deletion mutants (#1, #2, #3, and #4). The domains include the ATP-binding domain (residues 251–442), a helicase C-terminal domain (residues 459–716), a helicase-associated domain (residues 736–859) and an oligonucleotide domain (residues 925–1010). F, HEK293T cells were cotransfected with the indicated HA-tagged PLRG1 constructs along with plasmids encoding Flag-tagged DHX37, and the interaction between DHX37 and PLRG1 was evaluated by IP and immunoblotting. G, Schematic representation of HA-tagged full-length PLRG1 (FL) along with its various deletion mutants (N-terminal or WD repeat deletion mutants). H, HEK293T cells were cotransfected with the indicated Flag-tagged DHX37 constructs along with plasmids encoding HA-tagged PLRG1, and the interaction between DHX37 and PLRG1 was evaluated by IP and immunoblotting.

DHX37 interacts with PLRG1, a core component of the CDC5L complex, in liver cancer cells. A, DHX37-associated proteins were analyzed with STRING. The network nodes represent potential interacting proteins obtained by Co-IP/LC/MS. The edges represent protein–protein interactions. B, Co-IP using control IgG and an anti-Flag (DHX37) or anti-HA (PLRG1) antibody was carried out using extracts prepared from the HEK293T cells transfected with the pCMV-Flag-DHX37 and pCMV-HA-PLRG1 plasmids. The presence of DHX37 or PLRG1 in these immunoprecipitates was evaluated by immunoblot (WB) analysis. C, Co-IP using control IgG and an anti-DHX37 or anti-PLRG1 antibody was carried out using extracts prepared from Huh7 cells. The endogenous interaction of PLRG1 with DHX37 was evaluated by Western blot analysis with the corresponding antibodies. D, Huh7 and HepG2-C3A cells stably expressing DHX37 were fixed for immunofluorescence analysis. DHX37 was detected using an anti-Flag (DHX37) primary antibody and an Alexa Fluor 488 goat anti-mouse secondary antibody, and PLRG1 was detected using an anti-PLRG1 primary antibody and an Alexa Fluor 594 goat anti-rabbit secondary antibody. Representative cells from the same field for each experimental group are shown. Scale bars, Huh7, 50 µm (first column) or 25 µm (other four columns). HepG2-C3A, 25 µm (first column) or 10 µm (other four columns). E, Schematic representation of Flag-tagged full-length DHX37 (FL) along with its various deletion mutants (#1, #2, #3, and #4). The domains include the ATP-binding domain (residues 251–442), a helicase C-terminal domain (residues 459–716), a helicase-associated domain (residues 736–859), and an oligonucleotide domain (residues 925–1010). F, HEK293T cells were cotransfected with the indicated HA-tagged PLRG1 constructs along with plasmids encoding Flag-tagged DHX37, and the interaction between DHX37 and PLRG1 was evaluated by IP and immunoblotting. G, Schematic representation of HA-tagged full-length PLRG1 (FL) along with its various deletion mutants (N-terminal or WD repeat deletion mutants). H, HEK293T cells were cotransfected with the indicated Flag-tagged DHX37 constructs along with plasmids encoding HA-tagged PLRG1, and the interaction between DHX37 and PLRG1 was evaluated by IP and immunoblotting.