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R.G. Morgan et al.
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Figure 7. Modulation of LEF-1 expression affects downstream Wnt signaling. (A) Representative flow cytometric histograms showing intensity of the TCF reporter (BAR) in K562 and HEL cells treated with control/LEF1 shRNA ± CHIR99021. (B) Summary data showing the median fluorescence intensity generated from the BAR reporter in K562 and HEL cells treated with control/LEF1 shRNA ± CHIR99021. (C) Representative immunoblots showing expression of known Wnt target proteins survivin, c-MYC and cyclinD1 in ML-1 and U937 cells in response to control/LEF1 overexpression ± CHIR99021. Lamin A/C and a-tubulin were used to assess fraction purity and protein loading. (D) Summary data showing the relative fold-change in nuclear protein expression of classic Wnt targets survivin, c-MYC and cyclinD1 in CHIR99021 treated ML1 and U937 cells over-expressing LEF1. Dashed line represents relative level (=1) present in nuclei of CHIR99021-treated ML1 and U937 cells expressing control plasmid. *P<0.05; **P<0.01; ***P<0.001. ns: not significant.
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Figure 8. Short-form LEF-1 is proteolytically-derived and is capable of β-catenin binding. (A) Representative immunoblot showing total β-catenin co-immunoprecipi- tation (Co-IP) from cytosolic and nuclear fraction of LEF1-over-expressing U937 cells. Total β-catenin and LEF-1 protein levels are shown. ID: immunodepleted lysate. Representative immunoblot showing LEF-1 protein levels in (B) K562 control and (C) U937-LEF1 nuclear lysates ± protease inhibitor cocktail (PIC) during time course incubation at 37°C. (D) Representative immunoblots showing LEF-1 protein levels in K562 control nuclear lysate after mixing with U937 control whole cell lysate (1:1 protein concentration) ± PIC during time course incubation at 37°C. Nuclear lamin A/C detection was used to assess protein loading.
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haematologica | 2019; 104(7)