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. 2022 Jun 21;42(7):e00071-22. doi: 10.1128/mcb.00071-22

FIG 1.

FIG 1

OtDUB associates directly with adapter protein-1 and -2 (AP-1 and AP-2). (A) HeLa cell lysates treated with 30 mM N-ethylmaleimide (NEM) (+/−) were incubated with anti-Flag-resin or immobilized OtDUB-Flag resin. Input lysates and eluates were tested for putative binding partners by SDS-PAGE and immunoblotting (n = 1). Arrowhead denotes monoubiquitin (Ub1), UbN, ubiquitin conjugates. Numbers on the left in this panel and elsewhere indicate molecular mass markers (in kDa). (B and C) Superose 6 gel filtration chromatograms of purified proteins: AP2 core alone (green), OtDUB alone (black), or the complex of both (blue) (n = 2) (B); AP1 core alone (green), OtDUB alone (black), or the complex of both (blue) (n = 1) (C). (D) C-terminal truncations of GST-OtDUB or GST alone evaluated for enrichment by glutathione-resin pulldowns from bacterial lysates spiked with purified AP-1 or AP-2 core (n = 1). Eluates were resolved by SDS-PAGE and analyzed by immunoblotting or Ponceau-S staining (PonS). (E) Pulldown performed as in panel D with OtDUB250–450 and OtDUB275–675 being tested compared to controls (n = 1). For both D and E, representative Ponceau-S stains are used for the AP-1 and AP-2 pulldowns. (F, G, and H) Superose 6 gel filtration chromatograms of purified protein complexes. (F) AP2 core alone (green), OtDUB275–675 alone (black dashed), or the complex of both (blue) (n = 1). (G) OtDUB alone (black) or in complex with AP-2-μ2CTD-truncated (blue) (n = 1). (H) AP2 μ2CTD alone (green), OtDUB alone (black), or (blue) (n = 1). The same representative OtDUB alone chromatogram is used in panels B, C, G, and H. (I) Cartoon representation of the OtDUB fragments used in panel J. (J) Ectopically expressed Flag-tagged OtDUB fragments were immunoprecipitated from HeLa cell lysates, resolved by SDS-PAGE with input lysates and immunoblotted for AP-1 (AP1G1) and AP-2 (AP2M1) subunits and the positive control Rac1 (n = 2). mAU, mean arbitrary units; FL, full length; GEF, guanine nucleotide exchange factor; UBD, ubiquitin-binding domain.