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. 2022 Sep 20;41(21):e110372. doi: 10.15252/embj.2021110372

Figure 3. TSG101 controls PARylation.

Figure 3

  • A
    Nuclear‐cytoplasmic fractionation of U2‐OS cells. Indicated samples were transfected with siRNAs against TSG101. PARylation was inhibited by olaparib (10 μM, 24 h before analysis). Cytoplasmic export of p‐ATM was induced by etoposide (50 μM, 45 min before analysis). Nuclear and cytoplasmic fractions were immunoblotted with the indicated antibodies. The fractionation efficiency was controlled by the respective subcellular marker proteins (PARP1 and LDH‐A). PARylation was detected using the Pan ADPr reagent. Note that PARylation is activated by shearing forces during extract preparation (lane 1) (Jungmichel et al, 2013).
  • B
    U2‐OS cells were transfected with nontargeting (−) or TSG101‐targeting (+) siRNAs and cells were irradiated (+) or not (−) as indicated (20 Gy) 10 min. Before analysis. Whole‐cell extracts were immunoblotted with the 10H PAR monoclonal antibody.
  • C
    siRNA‐transfected U2‐OS cells as in B were etoposide‐treated or not, as indicated. Whole‐cell extracts were immunoblotted with the Pan ADPr reagent that recognizes both poly‐ and mono‐(ADP‐ribose). Note that PARylation also occurred during cell lysis in untreated cells.
  • D
    U2‐OS cells were transfected with nontargeting or VPS28‐targeting siRNAs. Whole‐cell extracts were immunoblotted with indicated antibodies.
  • E
    Total mRNA was extracted from the samples in D. Expression of TSG101 was analyzed by RT–qPCR and normalized to two housekeeping genes, ACTA1 and RPL13A. The conditions were compared with an ordinary one‐way ANOVA (ns, P > 0.05). Error bars represent mean ± SEM.
  • F
    Cells were irradiated (20 Gy) as indicated 10 min before analysis. Whole‐cell extracts were immunoblotted with the indicated antibodies.

Data information: The results are each presentative for three (panels A–D and F) or six (panel E) independent experiments.