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. 2022 Mar 29;41(9):e110145. doi: 10.15252/embj.2021110145

Figure 3. K27‐linked ubiquitylation predominantly occurs in the nucleus and impacts the integrity of the p97 system.

Figure 3

  • A
    Immunoblot analysis of Ub‐K27 antibody reactivity with recombinant linkage‐specific di‐Ub proteins.
  • B
    Representative images of U2OS/shUb/HA‐Ub(WT) or U2OS/shUb/HA‐Ub(K27R) cells immunostained with antibody to K27‐linked Ub (Ub‐K27). Scale bar, 10 μm.
  • C
    Intensity of nuclear signal recognized by Ub‐K27 antibody in U2OS/shUb/HA‐Ub(K27R) cells treated with DOX for the indicated times was quantified using QIBC (solid lines, median; dashed lines, quartiles (a.u., arbitrary units); > 3,000 cells analyzed per condition).
  • D
    Schematic depicting experimental setup for isolation and quantitative label‐free mass spectrometry analysis of HA‐Ub conjugates isolated from whole cell lysates of DOX‐treated Ub(WT) and Ub(K27R)‐c21 replacement cell lines by HA immunoprecipitation (IP) under denaturing conditions.
  • E
    Global ubiquitylation changes induced by Ub(K27R) replacement, profiled by mass spectrometry analysis of HA‐Ub conjugates isolated as shown in (D) (see also Fig EV3C and D; for full results see Dataset EV1). Volcano plot shows enrichment of individual proteins (HA‐Ub(WT)/HA‐Ub(K27R) ratio) plotted against the P value. Significance thresholds (FDR < 0.05; s0  = 1) are indicated. Nuclear proteins with a known interplay with p97 are highlighted in red. See Fig 1I for Ub di‐Gly peptide quantification.
  • F, G
    HA‐Ub conjugates isolated as in (D) were immunoblotted with indicated antibodies. See also Fig EV3F.
  • H
    DOX‐treated Ub replacement cell lines treated with p97i for the indicated times were fixed and immunostained with Ub conjugate‐specific antibody (FK2). Levels of Ub conjugates in the nucleus were quantified using QIBC (solid lines, median; dashed lines, quartiles; > 2,500 cells analyzed per condition).
  • I
    As in (H), except cells were subjected to stringent pre‐extraction before fixation (solid lines, median; dashed lines, quartiles; > 5,000 cells analyzed per condition).
  • J
    Uninduced U2OS/shUb/HA‐Ub cell lines treated or not with p97i for 8 h were analyzed as in (H) (solid lines, median; dashed lines, quartiles; > 5,000 cells analyzed per condition).
  • K
    As in (H), except cells were immunostained with CHOP antibody (solid lines, median; dashed lines, quartiles (a.u., arbitrary units); > 3,000 cells analyzed per condition).

Data information: Data (A‐C,F‐K) are representative of three independent experiments with similar outcome.