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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: Cancer Discov. 2020 May 22;10(9):1388–1409. doi: 10.1158/2159-8290.CD-19-1436

Fig. 2. Posttranslational regulation of SRSF6 by USP7.

Fig. 2.

A, Immunoblot showing SRSF6 protein levels in normal CD4+ T-cells (n=2) and CD3+ T cells (n=2), T-ALL patients (n=7), and CUTLL1 and JURKAT cells. B, C, Quantification of immunoblot bands presented in A. USP7 and SRSF6 protein levels are higher in T-ALL compared to T cells (B). USP7 protein levels significantly correlate with SRSF6 levels in T-ALL (C). Actin is used as a loading control. D, E, RPPA analysis for SRSF6 protein levels in HR (n=16) vs. non-HR T-ALL (n=31) cases (D) and correlation with USP7 expression (E). F, USP7 immunoprecipitation coupled to mass spectrometry (IP-MS) analysis in JURKAT cells. Shown is the overlap of USP7-associated proteins across 3 biological replicates, revealing splicing factors associated with USP7. G, Schematic representation of the USP7-related lysine ubiquitome analysis in JURKAT cells. H, Network analysis of the overlapped proteins of USP7 immunoprecipitation-mass spectrometry and KGG mass spectrometry using GeneMANIA. Splicing related proteins are highlighted in red. I, Analysis of the overlapping data sets for USP7 immunoprecipitation-mass spectrometry and KGG mass spectrometry studies reveals a significant number of RNA binding proteins in common (25, P< 0.0001). J, Immunoblot for detection of ubiquitination upon lentiviral expression of Flag-tagged SRSF6 in CUTLL1 cells coupled to treatment with P5091. The Flag epitope was used for SRSF6 pulldown. A representative blot for one biological replicate of vehicle- and P5091-treated CUTLL1 cells for the pull-down is shown. K, Immunoblots (WB) following immunoprecipitation (IP) of USP7 (left panel) and SRSF6 (right panel) in JURKAT cells, showing interaction of USP7 and SRSF6. L, Immunoblot studies for USP7 and SRSF6 using CUTLL1 and JURKAT cells upon treatment with increasing concentrations of P5091. Actin is used as a loading control. M, Immunoblot studies for USP7 and SRSF6 upon silencing of USP7 using two different short-hairpin RNAs in CUTLL1 cells (left panel) or siRNA over a period of 96h in 293T cells (right panel). Actin is used as a loading control. N, Immunoblot analysis for SRSF6 levels upon treatment with cycloheximide (CHX, 200μg/ml), or combination of CHX with P5091 (10μM) over a period of 24h. Representative blot from one out of three experiments (left) and quantification of protein levels from three experiments (right) are shown (***, P<0.001).