American Society of Hematology

Figure 5.

$Wdr26 regulates lamin B ubiquitination in differentiating erythroblasts. (A-B) The ubiquitination level in the cytoplasm and nucleus of the chemically induced wild-type, (A) Wdr26-knockout, or (B) Rmnd5a-knockout MEL cells. The proteasome inhibitor MG132 (2.5 μM) was used to inhibit the degradation of ubiquitinated proteins. W, wild type; K, (A) Wdr26-knockout or (B) Rmnd5a-knockout. (C) Ubiquitination of Lamin B was alleviated in Wdr26-knockout MEL cells. *Lamin B. (D) Decreased rate of Lamin B degradation in Wdr26-knockout MEL cells. CHX (100 μg/mL) was used to inhibit protein synthesis. *Cleaved Lamin B. (E) Quantitative analysis of relative Lamin B protein level in panel D. Error bars represent SEM from 3 replicates. **P < .01. The Lamin B level in indicated time points was normalized to 0 hours. (F) Western analysis of Lamin B in DMSO-induced MEL cells treated with the protein synthesis inhibitor cycloheximide (CHX, 100 μg/mL) in combination with the proteasome inhibitor MG132 (2.5 μM) or the vacuolar H+ ATPase inhibitor bafilomycin A1 (Baf A1, 100 nM). (G) Treatment with leptomycin B (60 nM) did not alter the ubiquitination level of Lamin B in MEL cells. *Lamin B. (H) Ubiquitination of Lamin B by Wdr26-FLAG pull-down (PD) fraction in vitro. (I) Ubiquitination of Lamin B by Ranbp10-FLAG or Gid8-FLAG PD fractions derived from wild-type (with Wdr26) or Wdr26-konckout (without Wdr26) MEL cells. (J) Lysates from HEK293 cells transfected with FLAG-Wdr26 and HA-Lamin B were immunoprecipitated with anti-FLAG antibody or immunoglobulin G, and then immunoblotted with anti-HA or anti-FLAG antibodies (left). Ectopically expressed FLAG-Wdr26 was immunoprecipitated by endogenous Lamin B in HEK293 cells (right). *FLAG-Wdr26 (left) or Lamin B (right). (K) The N-terminal CTLH domain of Wdr26 interacts with Lamin B. HEK293 cells were transfected with FLAG-Wdr26-N (1-302 aa) or FLAG-Wdr26-C (303-641 aa). Immunoprecipitates by endogenous Lamin B were analyzed by immunoblotting with anti-FLAG antibodies. *Lamin B. (L) The C-terminal tail region of Lamin B interacts with Wdr26. The HA-tagged Lamin B-N (1-45 aa), Lamin B-CR (36-397 aa), or Lamin B-C (398-615 aa) constructs were transfected into HEK293 cells together with FLAG-Wdr26. The cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted using anti-HA antibody. *FLAG-Wdr26. (M) Immunoblotting analysis of lamin B in the peripheral blood of wild-type and wdr26b−/− fish.$

Wdr26 regulates lamin B ubiquitination in differentiating erythroblasts. (A-B) The ubiquitination level in the cytoplasm and nucleus of the chemically induced wild-type, (A) Wdr26-knockout, or (B) Rmnd5a-knockout MEL cells. The proteasome inhibitor MG132 (2.5 μM) was used to inhibit the degradation of ubiquitinated proteins. W, wild type; K, (A) Wdr26-knockout or (B) Rmnd5a-knockout. (C) Ubiquitination of Lamin B was alleviated in Wdr26-knockout MEL cells. *Lamin B. (D) Decreased rate of Lamin B degradation in Wdr26-knockout MEL cells. CHX (100 μg/mL) was used to inhibit protein synthesis. *Cleaved Lamin B. (E) Quantitative analysis of relative Lamin B protein level in panel D. Error bars represent SEM from 3 replicates. **P < .01. The Lamin B level in indicated time points was normalized to 0 hours. (F) Western analysis of Lamin B in DMSO-induced MEL cells treated with the protein synthesis inhibitor cycloheximide (CHX, 100 μg/mL) in combination with the proteasome inhibitor MG132 (2.5 μM) or the vacuolar H⁺ ATPase inhibitor bafilomycin A1 (Baf A1, 100 nM). (G) Treatment with leptomycin B (60 nM) did not alter the ubiquitination level of Lamin B in MEL cells. *Lamin B. (H) Ubiquitination of Lamin B by Wdr26-FLAG pull-down (PD) fraction in vitro. (I) Ubiquitination of Lamin B by Ranbp10-FLAG or Gid8-FLAG PD fractions derived from wild-type (with Wdr26) or Wdr26-konckout (without Wdr26) MEL cells. (J) Lysates from HEK293 cells transfected with FLAG-Wdr26 and HA-Lamin B were immunoprecipitated with anti-FLAG antibody or immunoglobulin G, and then immunoblotted with anti-HA or anti-FLAG antibodies (left). Ectopically expressed FLAG-Wdr26 was immunoprecipitated by endogenous Lamin B in HEK293 cells (right). *FLAG-Wdr26 (left) or Lamin B (right). (K) The N-terminal CTLH domain of Wdr26 interacts with Lamin B. HEK293 cells were transfected with FLAG-Wdr26-N (1-302 aa) or FLAG-Wdr26-C (303-641 aa). Immunoprecipitates by endogenous Lamin B were analyzed by immunoblotting with anti-FLAG antibodies. *Lamin B. (L) The C-terminal tail region of Lamin B interacts with Wdr26. The HA-tagged Lamin B-N (1-45 aa), Lamin B-CR (36-397 aa), or Lamin B-C (398-615 aa) constructs were transfected into HEK293 cells together with FLAG-Wdr26. The cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted using anti-HA antibody. *FLAG-Wdr26. (M) Immunoblotting analysis of lamin B in the peripheral blood of wild-type and wdr26b^−/− fish.

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