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. 2013 Dec;25(12):2511–2517. doi: 10.1016/j.cellsig.2013.08.013

Fig. 2.

Fig. 2

CHFR ubiquitinates and degrades TOPK. (a) Myc tagged full length CHFR, ΔRING or ΔCysteine rich domain CHFR mutants were expressed in HeLa cells along with triple tagged TOPK and HA tagged ubiquitin. After the 24 hour post transfection, cells were treated with MG132 (10 μM) for 6 h and the levels of TOPK ubiquitination were evaluated by immunoprecipitation using streptavidin beads followed by anti-HA immunoblotting. (b) HeLa cells were transiently transfected with TOPK or TOPK D4 constructs along with or without CHFR and TOPK ubiquitination was evaluated by immunoprecipitation using streptavidin beads followed by anti-ubiquitin immunoblotting. (c) HeLa cells transfected with indicated vectors were treated with cyclohexamide and the lysates were collected at the indicated time points. The levels of TOPK, CHFR and actin were determined by immunobloting. (d) HeLa cells were transfected with TOPK along with vector or CHFR and were left untreated or treated with MG132. Cells were treated with cyclohexamide and the lysates were collected at the indicated time points. The levels of TOPK, CHFR and actin were determined by immunobloting.