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. Author manuscript; available in PMC: 2020 Apr 15.
Published in final edited form as: Cancer Res. 2019 Feb 1;79(8):1913–1924. doi: 10.1158/0008-5472.CAN-18-3037

Figure 5. The OTUB1-SLC7A11 interaction is tightly regulated by CD44 in human cancer cells.

Figure 5.

A. Western Blot analysis for CD44 and SLC7A11 from H1299 cells transfected with control siRNA or a pool of CD44-specific siRNAs.

B. H1299 cells transfected with control siRNA or a pool of CD44 siRNAs and then treated with TBH (40 μM) and Ferr-1 (2 μM) as indicated for 6 hours. Quantification of cell death from three technical replicates is shown; error bars represent the mean ±s.d.

C. Western Blot analysis for SLC7A11, OTUB1 and CD44 from H1299 cells transfected with HA-SLC7A11 and either an empty vector or increasing amounts of a Flag-CD44 expressing vector.

D. Western Blot analysis for SLC7A11, OTUB1 and CD44 from T24 control cells and OTUB1-null cells transfected with HA-SLC7A11 and either an empty vector or increasing amounts of a CD44-expressing vector as indicated.

E. Western Blot analysis for HA-SLC7A11, OTUB1 and CD44 from H1299 control cells and OTUB1 CRISPR cells transfected with HA-SLC7A11 and either an empty vector or a CD44-expressing vector.

F. Western Blot analysis for OTUB1 after immunoprecipitation of SLC7A11, with anti-HA antibody-coupled beads, from HA-SLC7A11 stable H1299 cells transfected with CD44 and Flag-OTUB1 as indicated. The amount of SLC7A11 was normalized after immunoprecipitation. 1% of the sample was loaded as input.

G. Western Blot analysis for OTUB1 after immunoprecipitation of Flag-SLC7A11, with anti-Flag (M2) antibody-coupled beads, from H1299 cells transfected with control siRNA or a pool of CD44-specific siRNAs, Flag-SLC7A11 and OTUB1 individually or together as indicated. The amount of SLC7A11 was normalized after immunoprecipitation. 1% of the sample was loaded as input.