Figure 4. MYCBP2 loss-of-function increases EPHB2 protein turnover in HeLa cells.

(A) Induced EPHB2-FLAG expression is reduced in MYCBP2^CRISPR HeLa cells. (B) Western blotting for transfected EPHB2-FLAG in CTRL^CRISPR and MYCBP2^CRISPR cells. (C) Quantification of transfected EPHB2-FLAG levels (p=0.0046, one-sample t-test). (D) Representative western blot of EPHB2-FLAG in CTRL^CRISPR and MYCBP2^CRISPR cells treated with DMSO or cycloheximide for 4 hr and 8 hr. (E) Quantification of EPHB2-FLAG turnover with cycloheximide (CTRL^CRISPR vs. MYCBP2^CRISPR at 8 hr eB2-Fc stimulation, p=0.0474, two-way ANOVA followed by Tukey’s multiple comparison test). (F) Western blot showing EPHB2-FLAG degradation when cells are challenged with ephrin-B2 (1 µg/ml) for different periods of time. (G) Quantification of ephrin-B2-evoked EPHB2 degradation (ns, not significant; two-way ANOVA followed by Tukey’s multiple comparison test). Although not significant, there is an apparent trend towards lower EPHB2 levels in MYCBP2^CRISPR cells, which could become significant with additional replicates. (H) Western blot of EPHB2 ubiquitination in CTRL^CRISPR and MYCBP2^CRISPR cells. (I) Quantification of ubiquitinated EPHB2. CTRL^CRISPR cells stimulated with Fc vs. eB2-Fc, p=0.1349 (One sample t-test); MYCBP2^CRISPR cells stimulated with Fc vs. EB2-Fc, p=0.0195 (Unpaired two-tailed t-test). (J) After tetracycline induction of EPHB2-FLAG expression for 16 hr, CTRL^CRISPR and MYCBP2^CRISPR HeLa cells were treated with DMSO (1:500) or inhibitors of the proteasome (MG132 50 µM) or lysosome (BafA1 0.2 µM; CoQ 50 µM) for 6 hr, and EPHB2 levels were analysed by western blotting. (K) Quantification of EPHB2 levels following treatment with proteasome or lysosome inhibitors. Statistical significance for the comparison between CTRL^CRISPR cells treated with DMSO or inhibitors was determined by one-sample t-test (MG132, p=0.0598; BafA1, p=0.0200; CoQ, p=0.3632), whereas statistical significance for the comparison between MYCBP2^CRISPR cells treated with DMSO and individual inhibitors was determined by two-tailed paired t-test (MG132, p=0.8893; BafA1, p=0.0361; CoQ, p=0.0835). (L) GFP-EPHB1 and HA-EPHB3 transfected into CTRL^CRISPR and MYCBP2^CRISPR HeLa cells and detected by western blot. (M) Quantification of GFP-EPHB1 and HA-EPHB3 levels (EPHB1, p=0.0588; EPHB3, p=0.4253; one-sample t-test). (N) FLAG-EPHA3 transfected into CTRL^CRISPR and MYCBP2^CRISPR HeLa cells and detected by WB. (O) Quantification of FLAG-EPHA3 (p=0.5369, one-sample t-test). Error bars represent SD.