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. 2022 Dec 19;13(12):1053. doi: 10.1038/s41419-022-05500-6

Fig. 4. Akt/GSK3β signal pathway is essential for TRAF4 knockout-mediated MCL-1 degradation.

Fig. 4

A Immunoblotting for IP-mediated TRAF4 and Akt expression in CAL27R cells treated with/without IR (2 Gy) for 20 min. B CAL27R cells were co-transfected with Flag-TRAF4 and HA-Akt for 48 h, followed by IR (2 Gy) treated for 20 min. Immunoblotting was performed to analyze IP-mediated Akt ubiquitination. C 293T cells were co-transfected with Flag-TRAF4, HA-Akt and His-ubiquitin mutant (K6R, K11R, K27R, K29R, K33R, K48R, K63R, WT) for 48 h, followed by IR (2 Gy) treated for 20 min. Ni-NTA pull-down assay was performed to detect Akt ubiquitination. D 293T cells were co-transfected with Flag-TRAF4 WT or Flag-TRAF4 C18A mutant, HA-Akt and His-Ub for 48 h, followed by IR (2 Gy) treated for 20 min. Ni-NTA pull-down assay was performed to detect Akt ubiquitination. E Immunoblotting for IP-mediated Akt ubiquitination in TRAF4 knockout CAL27R cells treated with or without IR (2 Gy) for 20 min. F CAL27R cells were co-transfected with Flag-TRAF4, HA-Akt WT, or HA-Akt K8/K14R mutant and His-Ub for 48 h, followed by IR (2 Gy) treated for 20 min. Ni-NTA pull-down assay was performed to detect Akt ubiquitination. G TRAF4 knockout CAL27R and SCC25R cells were treated with/without IR (2 Gy) for 20 min, and WCE was subjected to immunoblotting analysis. H TRAF4 knockout CAL27R cells were treated with IR (2 Gy) for different time points, and WCE was subjected to immunoblotting analysis. I GSK3β depleted CAL27R and SCC25R cells were treated with IR (2 Gy) for 20 min, and WCE was subjected to immunoblotting analysis. J TRAF4 knockout CAL27R and SCC25R cells were treated with SB216763 inhibitor for 24 h, followed by IR (2 Gy) treated for 20 min. WCE was subjected to immunoblotting analysis.