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. 2023 May 11;108(10):2677–2689. doi: 10.3324/haematol.2022.282211

Figure 3.

Figure 3.

Aging-declined CRL4DCAF1-mediated ubiquitination degradation signaling leads to the increase of PUS10. (A) This histogram depicts the mRNA expression of Pus10 in young (2 months) and aged (31 months) hematopoietic stem and progenitor cells (HSPC). (B) Affinity purification of PUS10 protein from HEK293T cells stably expressing Flag-tagged PUS10. Proteins identified by mass spectrometry are listed. The bait protein is marked in bold letters. (C) HEK293T cells were co-transfected with plasmids encoding SFB-tagged PUS10 and Myc-tagged DDB1, DCAF1, CUL4B followed by co-immunoprecipitation using anti-Flag, anti-Myc antibody. Representative western blot (WB) shows that PUS10 interacts with DDB1, DCAF1 and CUL4B. (D) HEK293T cells were co-transfected with plasmids encoding SFB-tagged PUS10, Myc-tagged DDB1, DCAF1, CUL4B and HA-tagged Ub-WT or Ub-K48R followed by coimmunoprecipitation using anti-HA, anti-Flag, anti-Myc antibody. Representative WB shows that PUS10 is ubiquitinated by the CRL4DCAF1 complex. (E) Representative WB shows the expressions of DDB1 and CUL4B in young (3 months) and aged (28 months) HSPC. (F) 32D cells were infected by lentivirus carrying Ddb1-shRNA. 72 hours later, green fluorescent protein-positive (GFP+) cells were sorted for WB to validate the expression of PUS10 and DDB1. Representative WB shows the expression of PUS10 and DDB1 in vector and DDB1-KD 32D cells. (G) This histogram depicts the protein level of PUS10 in vector and DDB1-KD 32D cells from quantitative WB data (N=2). (H) This histogram depicts the protein expression of DDB1 in vector and DDB1-KD 32D cells from quantitative WB data (N=3).