Fig. 2: EIF3H knockdown decreases steady-state expression of HAX1 protein in CRC cells. | Nature Communications

Fig. 2: EIF3H knockdown decreases steady-state expression of HAX1 protein in CRC cells.

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

Fig. 2

a Coomassie blue staining of the immunoprecipitated profile using anti-Flag M2 beads in HCT116 cells transfected with empty vector or Flag-tagged EIF3H. The purified EIF3H protein complex was subjected to mass spectrometry analysis. b The interaction between exogenous and endogenous EIF3H and HAX1 was determined by co-IP assay. HEK293T cells were transfected with Flag-EIF3H or Flag-HAX1 plasmid. The cell lysates were pulled down with anti-Flag M2 beads and immunoblotted with the indicated antibodies. HCT116 cell lysates were immunoprecipitated with either control rabbit IgG, EIF3H, or HAX1 antibodies followed by immunoblotting. c Proximity ligation assay. A representative image series was shown. Red spots mark positive PLA signals for EIF3H-HAX1 interactions. Nuclei were stained with DAPI. Scale bar = 50 µm. d Immunoblot analysis of HAX1 expression in shCtrl and shEIF3H DLD1 and HCT116 cells. e shCtrl and shEIF3H CRC cells were treated with or without 25 μM MG132 for 6 h. Cell lysates were immunoblotted. f HEK293T cells transfected with the indicated plasmids were treated with cycloheximide (CHX) (100 μg/mL) for indicated time. Cell lysates were immunoblotted. HAX1 levels were quantified, normalized, and the turnover of HAX1 is indicated graphically in the right panel. g shCtrl and shEIF3H DLD1 and HCT116 cells were treated with CHX. HAX1 turnover rate was analyzed by immunoblotting (left) and quantified (right). h HEK293T cells were transfected with the indicated plasmids and treated with MG132. The cell lysates were pulled down with Ni-NTA beads and immunoblotted. i Cellular extracts from shCtrl and shEIF3H HCT116 cells were concentrated and fractionated on Superose 6 size exclusion columns. An equal volume from each chromatographic fraction was analyzed by western-blot. Chromatographic eluate profiles and molecular size of eluted fraction were indicated. j shEIF3H HCT116 cells were infected with lentivirus containing control vector or Flag-HAX1 vector. The indicated protein levels, cell proliferation, colony formation, migration, and invasion were indicated. The data are presented as the means ± SD. The p values were obtained by two-tailed unpaired t test. All data shown (including immunoblotting, immunofluorescent staining) were obtained from at least three biological independent experiments with similar results. Source data are provided as a Source Data file.

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