Research Paper Volume 15, Issue 19 pp 10593—10606
UCHL1-PKM2 axis dysregulation is associated with promoted proliferation and invasiveness of urothelial bladder cancer cells
Figure 5. UCHL1 upregulates the expression of PKM2 in UBC cells via de-ubiquitination modulation. (A–C) WB assay on protein samples from UBC cell line groups respectively transfected with UCHL1 overexpression vector or shRNAs, in combination with or without MG132, in order to explore the impact of UCHL1 modulation on PKM2. (D–I) CHX chase experiment to detect PKM2 protein level degradation in UBC cell lines treated with UCHL1 shRNAs or UCHL1 wildtype/mutated overexpression vectors (n = 3). (J, K) PKM2 ubiquitination level detection using CO-IP method in UBC cell line groups treated with UCHL1 shRNAs or UCHL1 wildtype/mutated overexpression vectors. (L) PKM2 ubiquitination level detection using CO-IP method in HEK-293T cell line groups exogenously transfected with escalating dosage of His-tagged UCHL1 vectors, in combination of Flag-tagged PKM2 vectors and HA-tagged ubiquitin. (M) PKM2 ubiquitination level detection using CO-IP method in HEK-293T cell line groups exogenously transfected with His-tagged wildtype or mutated UCHL1 vectors, or control vectors, in combination of Flag-tagged PKM2 vectors.