DKK1-CKAP4 axis activates NF-κB pathway thorough recruiting CAND1. (A) Levels of p-p65 and IκB-α in MM.1S cells bearing an ICD domain depletion CKAP4 (ΔICD) and treated with rhDKK1(100 ng/mL) for 12 hours. (B) Silver staining of lysate from HEK-293T cells transfected with vector or CKAP4-flag for 48 hours. (C) Coimmunoprecipitation (co-IP) assay shows interactions between exogenous CAND1-flag with CKAP4 or exogenous CKAP4-flag with CAND1 in HEK-293T cells. Input, 2% lysate. IP, M2-flag antibody. (D) Co-IP assay shows bilateral interactions between endogenous CAND1 and CKAP4 in MM.1S cells. (E) Alteration of interaction between CAND1 and CUL1 in HEK-293T cells transfected with vector control or CKAP4-flag for 48 hr and then treated with rhDKK1(100 ng/mL) for 12 hours. Input, 2% whole cell lysate. IP, anti-CUL1 antibody. (F) Immunofluorescence assay showing colocalization of CKAP4 with CAND1 in A549 cells treated with rhDKK1 (100 ng/mL) for 12 hours. Scale bar, 10 μm. (G) Levels of p-p65 and IκB-α in MM.1S and OPM-2 cells stably expressing NT Ctrl or CAND1-shRNA. (H) Ubiquitination status of IκB-α in HEK293T cells transfected with shCAND1 or NT Ctrl for 48 hours. (I) Degradation rate of IκB-α protein in MM.1S and OPM-2 cells with CAND1 knockdown and then treated with 20 μM cycloheximide for up to 12 hours. (J) Western blotting shows the cleavage of PARP as a marker of apoptosis in MM cells stably expressing NT Ctrl or CAND1-shRNA (CAND1 KD) treated with BTZ and rhDKK1, or (K) anti-DKK1 for 24 hours.