Figure 3.

STUB1 depletion increased stability of RUNX1. A, K562 cells were transduced with a vector control or two independent gRNAs targeting STUB1. The gRNAs showed efficient depletion of STUB1, which resulted in the increased expression of RUNX1. B, whole-cell extracts from K562 cells described in A were immunoprecipitated with anti-RUNX1 antibody, and ubiquitinated RUNX1 was detected with anti-ubiquitin. STUB1 depletion resulted in the reduced ubiquitination of RUNX1. C, K562 cells transduced with a vector or STUB1 gRNAs were treated with 25 μg/ml of CHX for the indicated times, and cell extracts were analyzed with anti-RUNX1 antibody. Band intensities of RUNX1 and GAPDH were quantified with Multi Gauge (Science Lab), and the intensities of RUNX1 relative to GAPDH are shown. The value of RUNX1/GAPDH without CHX treatment was set to 1. Representative data (upper) and cumulative data from three independent experiments (lower, mean ± S.E., **, p < 0.01; +, p < 0.05; ++, p < 0.01) are shown. IB, immunoblot.