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. 2019 May 31;116(25):12311–12320. doi: 10.1073/pnas.1902012116

Fig. 4.

Fig. 4.

Ubiquitylated FBXW7 is degraded by both proteasome and p62-mediated lysosome pathways. (A) Autophagy involvement in FBXW7 degradation. Paired MEF cells with Atg5-WT or KO were transfected with FLAG-LSD1, then switched 48 later to fresh medium containing CHX with or without MG132 and CQ treatment for indicated periods, and harvested for IB. (B and C) p62 binds to ubiquitylated FBXW7. Cells were transfected with indicated plasmids for 48 h and treated with or without MG132 and CQ in the last 6 h, lysates were subjected to IP with p62-Ab (B) or HA-Ab (C), and followed by IB. (D) p62 knockdown partially reverses LSD1 reduction of FBXW7. Cells were transfected with si-p62 or si-control for 48 h, then transfected with indicated plasmids for another 48 h, in the absence or presence of MG132 in the last 6 h before harvesting for IB. The blot was quantified by densitometry scan using ImageJ software. The values in the blots were calculated after normalization of β-actin with the control sample setting at 1.