anti ubiquitin  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: SplitTurboID mapping of dimeric protein phosphatase complex interactomes

doi: 10.1016/j.isci.2026.115195

Figure Lengend Snippet: c20orf27 is a novel PP1 interactor linked to mRNA splicing that also associates separately with an E3 ubiquitin ligase complex (A) Putative RVxF motif in wild-type c20orf27 and mutation of the hydrophobic residues that should abrogate PP1 binding. Homogenous distribution of GFP-c20orf27 throughout the cytoplasm in formaldehyde-fixed U2OS cells. (B) Biofluorescence complementation (BiFC) assays confirm the re-formation of functional YFP when its N- and C-terminal halves are tagged to wild-type c20orf27 and PP1. This association is lost for the KAGA mutant. For A-B, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision CORE system using a 60x/NA1.4 oil immersion objective. (C) Quantitative IP/MS comparing GFP-c20orf27 (H) to GFP (L) in HeLa cells identified significant enrichment (>2-fold) of PP1 and a CUL2 E3 ubiquitin ligase complex with c20orf27. (D) Validation of the co-immunoprecipitation of the ligase substrate-specifier APPBP2 with GFP-c20orf27. To assess the impact of proteasomal inhibition, the assay was carried out in both untreated cells and in cells treated with the proteasome inhibitor MG132 (10 μM for 1 h; confirmed by anti-ubiquitin staining of the lysates). Ponceau S staining of the membrane (right panel) confirmed equivalent total protein loading and immunoprecipitation. (E) Functionally grouped biological process networks generated using Cytoscape:ClueGO for high-confidence hits identified in a splitPP1:c20orf27 experiment overlapped with splitPP1:RRP1B and splitPP1:TPRN. (F) 2D heatmap graph compares the distribution of protein hits between the splitPP1:NIPP1 (green) and splitPP1:c20orf27 (magenta) datasets, based on their fractional enrichment in each. Arrows indicate the threshold of 0.75 above which proteins are considered to be enriched in a particular dataset. Lines indicate spliceosome components and hnRNP proteins that were enriched above threshold for splitPP1:NIPP1 and splitPP1:c20orf27, respectively (see Table in ). (G) Accumulation of proteins biotinylated by splitPP1:c20orf27 (magenta) at nuclear speckles (arrows) stained with anti-SC35-AF488 (green). U2OS cells co-expressing NTD-c20orf27 and CTD-PP1ɣ were incubated with 50 μM biotin for 4 h prior to formaldehyde fixation and streptavidin-AF647 staining. Enlarged panels show the individual anti-SC35-AF488 and biotin signals for the boxed region. (H) Localization of a pool of GFP-c20orf27 (green) at nuclear speckles (arrows) marked by anti-SC35-AF555 staining (magenta) in methanol-fixed U2OS cells. Enlarged panels show the individual GFP-c20orf27 and anti-SC35-AF555 signals for the boxed region. Additional accumulation of GFP-c20orf27 was observed at the perinuclear region (arrowhead). (I) Analysis of co-expressed mCh-c20orf27 and APPBP2-GFP in methanol-fixed U2OS cells shows that both are enriched at the perinuclear region (arrowheads), with additional accumulation in nuclear foci (arrows) observed for mCh-c20orf27. For G–I, DNA was stained with Hoechst 33342 (blue), scale bars are 5 μm, and images were acquired on a DeltaVision Elite system using a 60x/NA1.4 oil immersion objective. See also .

Article Snippet: Monoclonal mouse anti-Ubiquitin , Santa Cruz , Cat#sc-8017; RRID: AB_628423.

Techniques: Ubiquitin Proteomics, Mutagenesis, Binding Assay, Functional Assay, Staining, Protein-Protein interactions, Biomarker Discovery, Immunoprecipitation, Inhibition, Membrane, Generated, Expressing, Incubation