Fig 1: 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.
Fig 2: TRIB2 boosts PCBP2 and reduces the global K48-Ub level.a mRNA levels of PCBP2 in the control, Bel-7402, and SMMC-7721 cells with TRIB2 knocked down or overexpressed, as measured by qRT-PCR (n = 3). Data were analyzed by one-way ANOVA and expressed as mean ± SD. NS non-significance. b CHX chase experiments of PCBP2 in the control and Bel-7402 cells with or without TRIB2 knocked out in the presence or absence of TRIB2 (Twt) or TRIB2?D (T?D). The relative protein levels of PCBP2 were normalized to those of GAPDH, and the “0 h” point was arbitrarily set to 100%. Data were expressed as mean ± SD. c K48-ubiquitination, K63-ubiquitination and total-ubiquitination of PCBP2, as immunoprecipitated by anti-PCBP2 antibodies for the indicated treatment in Bel-7402 cells, and measured by anti-K48, anti-K63, and anti-total Ub antibodies. The relative K48-Ub, k63-Ub, and total-Ub of PCBP2 were normalized to those of PCBP2 in the PCBP2-IPs and indicated below the blots. d Schematic representation of the Ub K48 and K63 linkages. e, f Global K48-Ub level was specifically targeted by TRIB2 and PCBP2. K48-Ub and K63-Ub levels were evaluated by anti-K48 and anti-K63 Ub antibodies in the Bel-7402 cells with TRIB2 (e) or PCBP2 (f) knocked down or overexpressed and treated cells with DMSO or BTZ (100 nM, 24 h). The relative protein levels of the global K48-Ub and K63-Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (e, f). Images of all the immunoblots are representative of three independent experiments.
Fig 3: PCBP2 is essential for TRIB2 to regulate PSMB5 activity.a Venn diagram showing three biologically independent MS results from the Bel-7402 cell immunoprecipitation with anti-TRIB2 antibodies. The candidates were further screened by UniProt online software and the literature to identify potential ubiquitination-related proteins. b Representative Coomassie blue stain image of the Bel-7402 cell immunoprecipitates pulled down by anti-TRIB2 or IgG antibodies (n = 3). c Ub in the control and Bel-7402 cells with MYPT1, GRP78, PARP1, or PCBP2 knocked down. d Ub in the control and Bel-7402 cells overexpressing PCBP1, PCBP2, or PCBP3 with or without TRIB2 knocked down. e Reciprocal co-IP results of purified TRIB2 and PCBP2 proteins in vitro. TRIB1, TRIB3, PCBP1, and PCBP3 were parallel examined to exclude non-specificity. f Positive correlation between PCBP2 and TRIB2 in the liver cancer specimens. TMA was performed by IHC using anti-PCBP2 and anti-TRIB2 antibodies (n = 208). The data were analyzed by Spearman’s rank correlation. g The effects of TRIB2 on Ub level were PCBP2-dependent. Ub in the control and Bel-7402 cells with or without TRIB2 knocked out or overexpressed, in the presence or absence of PCBP2 knocking out. h PCBP2 regulated Ub via PSMB5. Ub in Bel-7402 cells with PCBP2 and PSMB5 knocked out or overexpressed, as indicated. i TRIB2 regulated PSMB5 activity via PCBP2. A proteasome activity assay kit (AAT Bioquest) was used to evaluate PSMB5 activity in the Bel-7402 and SMMC-7721 cells under the same treatment as indicated in g (n = 3). Data were analyzed by one-way ANOVA and expressed as mean ± SD. ****P < 0.0001; NS non-significance. Images of all the immunoblots are representative of three independent experiments. The relative protein levels of the conj & poly Ub and mono Ub were normalized to those of GAPDH as calculated by ImageJ software and indicated just below the blots (c, d, g, h).
Fig 4: CCT3 blocked the interaction between PCBP2 and YAP/TFCP2.a PCBP2 directly interacted with YAP but not with TFCP2, as evaluated by PLA in Bel-7402 and SMMC-7721 cells. Scale bar, 50 µm. b YAP, TFCP2 bond with PCBP2, as measured by co-IP in Bel-7402 cells. c Schematic presentation of the hypothesis that CCT3 might prevent YAP/TFCP2 from interaction with PCBP2. d Interaction between exogenous PCBP2 and YAP, but not between PCBP2 and TFCP2, was identified by co-IP in Bel-7402 cells. e The WB2 motif was essential for the interaction between PCBP2 and YAP, as measured by co-IP in Bel-7402 cells. d, deletion. f The PCBP2-YAP interaction replied on the two WW domains in YAP, as measured by co-IP in Bel-7402 cells. d, deletion. g The WB2 motif was critical for ubiquitination of YAP and TFCP2. h The WB2 motif of PCBP2 was essential for CCT3-mediated suppression of ubiquitination of YAP and TFCP2, as measured in Bel-7402 cells with or without expressing increasing concentrations of CCT3. i The two WW domains of YAP were prerequisite for suppression of TFCP2 by PCBP2, as measured in control cells, Bel-7402 cells with YAP knocked down with or without simultaneous expressing exogenous YAP-WT/YAP-d2WW. j The two WW domains of YAP were essential for CCT3-induced disassociation of PCBP2 from YAP/TFCP2, as measured by co-IP in Bel-7402 cells. To clearly identify the interaction between PCBP2, TFCP2 and YAP, we artificially adjusted the concentrations of PCBP2 to the same level in the immune precipitates that were immunoprecipitated by the anti-PCBP2 antibodies. Images of WB are representative of three independent experiments
Fig 5: PCBP2 and TRIB2 maintain cell viability via GPX4 under OS.a Spheroid formation by the Bel-7402 and SMMC-7721 cells with or without TRIB2 knocked out, in the presence or absence of overexpressed PCBP2 (n = 3); Scale bar = 30 µm. Data were analyzed by one-way ANOVA and expressed as mean ± SD. ***P < 0.001; ****P < 0.0001. b Tumor growth in athymic mice inoculated with Bel-7402 cells with or without TRIB2 knocked out in the presence or absence of overexpressed PCBP2 (n = 5/group); Scale bar = 4 mm. The data were analyzed by Student’s t-test and expressed as mean ± SD. ***P < 0.001; ****P < 0.0001. c–h Inhibition of GPX4 diminished the ability of TRIB2 and PCBP2 to protect cells against OS. Bel-7402 cells transfected with Dox-inducible shRNA targeting GPX4 (iGPX4sh1 (c, f); iGPX4sh2 (d, g)) were pretreated with or without Dox (700 ng/ml, 24 h) (c, d, f, g). Bel-7402 cells were also pretreated with or without RSL3 (5 µM) for 5 h (e, h). Then, the cells were exposed to the indicated concentration of t-BuOOH (c–e) or diquat (f–h) for another 24 h. Cell viability was determined and calculated as described in the “Materials and methods” section (n = 3). The data were analyzed by one-way ANOVA and expressed as mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001. NS non-significance.
Supplier Page from Abcam for Anti-PCBP2/hnRNP E2 antibody [EPR14858]