Fig. 5. The DQLVPD element and the KH3 domain are essential for TRIB2–PCBP2 interaction and function.

a Schematic representation of the construction of the PCBP2-expressing and TRIB2-expressing plasmids. b, c co-IP were performed between TRIB2 and PCBP2 and their mutants in HEK-293T cells as indicated. The asterisks indicate the specific bands that represent truncated versions of TRIB2 and PCBP2. M, indicates protein ladder. d The DQLVPD element and the KH3 domain were essential for TRIB2–PCBP2 binding. co-IP were performed between indicated TRIB2 and PCBP2 constructs in HEK-293T cells. e Direct TRIB2-PCBP2 interaction relies on the DQLVPD element and KH3 domain. Interactions were measured by PLA experiments with Bel-7402 cells expressing the TRIB2 and PCBP2 as indicated (n = 3); Scale bar = 25 µm. The average PLA signals per cell were graphed below. Data were analyzed by Student’s t-test and expressed as mean ± SD. ****P < 0.0001. f, g The TRIB2 DQLVPD element and PCBP2 KH3 domain were essential to reduce K48-Ub levels. K48-Ub and total-Ub levels were measured by anti-K48-Ub and anti-Ub antibodies in the control and Bel-7402 cells overexpressing TRIB2 (Twt or TΔD) (f) or PCBP2 (Pwt or PΔK) (g) in the treatment of DMSO, BTZ (100 nM, 24 h) or PSMB5 knockout. The relative protein levels of global K48-Ub, conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (f, g). Images of all the immunoblots are representative of three independent experiments.