Fig. 3: E3 ubiquitin ligase βTrCP promotes HAX1 ubiquitination through binding phosphodegron of HAX1. | Nature Communications

Fig. 3: E3 ubiquitin ligase βTrCP promotes HAX1 ubiquitination through binding phosphodegron of HAX1.

From: The EIF3H-HAX1 axis increases RAF-MEK-ERK signaling activity to promote colorectal cancer progression

Fig. 3

a 293 T cells were transfected with Flag-tagged HAX1 or Flag-tagged βTrCP. Cell lysates were pulled down with anti-Flag M2 beads and immunoblotted. b HCT116 cell lysates were pulled down with an anti-HAX1 or anti-βTrCP antibody and immunoblotted with βTrCP or HAX1. c Representative immunoblots showing HAX1 steady-state expression in 293 T and HCT116 cells upon βTrCP overexpression. d HCT116 cells transfected with either Flag-βTrCP or vector were treated with or without MG132. Lysates were immunoblotted with indicated antibodies. e HCT116 cells transfected with either WT Flag-βTrCP or Flag-△F-box-βTrCP were treated with CHX for the indicated times. The cell lysates were immunoblotted (left). The turnover rate of HAX1 was shown (right). f Representative immunoblots showing HAX1 steady-state expression in HCT116 cells upon △F-box-βTrCP overexpression. g 293T cells were cotransfected with Flag-HAX1 with or without Myc-βTrCP plus His-ubiquitin wild type (WT), K48R mutant or K63R mutant. Cells were treated with MG132 for 6 h before harvesting. The ubiquitinated HAX1 proteins were pulled down by Ni-NTA-agarose beads and detected with anti-Flag antibody. h Sequence alignment of the putative βTrCP-recognized degron on HAX1. 293 T cells were transfected with WT or mutant Flag-tagged HAX1 (2AA, 2DD). Location of S231 and T235 of HAX1 is indicated. The cell lysates were pulled down with anti-Flag M2 beads and immunoblotted. i Representative immunoblots showing the turnover rate of HA-tagged WT or dot mutant HAX1, with or without Myc-βTrCP overexpression in HCT116 cells (left). Quantification of HAX1 turnover rate by the Image J software (right). j, k 293 T cells were cotransfected with the indicated plasmids. Cells were treated with MG132 for 6 h before harvesting. The ubiquitinated HAX1 proteins were pulled down by Ni-NTA beads and detected with anti-Flag antibody. Representative immunoblots shown in figures were repeated three times independently with similar results. Source data are provided as a Source Data file.

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