Figure 2. UBL5 preferentially associates with spliceosomal proteins in human cells.

A Whole-cell extracts (WCE) of stable HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) for 48 h were analyzed by immunoblotting with antibodies to HA and MCM6 (loading control).

B HeLa/Strep-HA-UBL5 cells treated as in (A) were fixed and immunostained with HA antibody. Scale bar, 10 μm.

C Mass spectrometry (MS)-based analysis of UBL5-interacting proteins. HeLa cells expressing Strep-HA-UBL5 or not were cultured in heavy (H) or light (L) SILAC medium, respectively. Strep-HA-UBL5 and associated proteins enriched on Strep-Tactin Sepharose were analyzed by MS. Plot shows Z-scores (from SILAC H/L ratios) and total intensity of identified proteins. Selected prospective UBL5 interactors associated with the spliceosome are highlighted. See Supplementary Table S1 for full results.

D Selected proteins with high SILAC (H/L) ratios identified in the experiment in (C).

E Functional interactions of all proteins with a Z-score above 2.5 in the UBL5 pull-down experiment were obtained from the STRING database and visualized as a network. Node size corresponds to the Z-score. Known spliceosome components are highlighted in red.

F Gene ontology (GO) enrichment analysis of putative UBL5-interacting proteins. Significance of the enrichment is indicated for each term.

G Extracts of stable HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) for 48 h were subjected to Strep-Tactin pull-down followed by immunoblotting with indicated antibodies.