Figure 6 from E3 Ubiquitin Ligase Mind Bomb 1 is a Novel Negative Regulator of Tumor-Suppressive BMP Signaling

The ligase domains of MIB1 are essential for its regulatory function in BMP. A, Top, schematic (modified from (20)) of the endogenous MIB1 coding region, with exons shown as boxes and protein domains indicated. A, Bottom, Cas9-resistant MIB1 cDNA constructs, containing mutated PAM sequences (asterisks), express either WT MIB1 cDNA (MIB1 WT, blue) or MIB1 cDNA with all three RING domains deleted (MIB1ΔR1-3, orange). B, Immunoblot analysis of MIB1 protein levels in MIB1-iKO cells that were engineered to stably express Cas9-resistant MIB1 WT or MIB1 ΔR1-3 after 3 days of Dox (0.5 μg/mL) treatment. β-Actin served as the loading control. C–E, MIB1-iKO cells overexpressing MIB1 WT or MIB1ΔR1–3 were treated with Dox (0.5 μg/mL) for the indicated time points. C, Colony formation assay after 14 days of Dox treatment; scale bars, 10 mm. Images are representative of three independent experiments. D, CellTiter-Glo assay after 7 days of Dox treatment. Data represent three independent experiments. Two-way ANOVA with Tukey test; *, P < 0.05. “ns” denotes nonsignificant differences between no MIB1 cDNA cells and those expressing MIB1 constructs. E, Immunoblot analysis of BMPR2 immunoprecipitates from MIB1-iKO cells. Lane labels indicate MIB1 WT (no cDNA, −Dox), MIB1 KO (no cDNA, +Dox; Dox-induced KO of endogenous MIB1), MIB1 WT OE (MIB1-mNeonGreen expressed in MIB1 KO cells), and MIB1ΔR1-3 OE (MIB1ΔR1-3-IRES-mNeonGreen expressed in MIB1 KO cells). Cell lysates were subjected to IP with anti-BMPR2 antibody or IgG control, followed by IB with the indicated antibodies. Input lysates are shown for comparison. A longer exposure of the MIB1 immunoblot is included to facilitate detection of MIB1 in BMPR2 immunoprecipitates. Blue and orange arrowheads indicate BMPR2-associated MIB1 WT OE and MIB1ΔR1-3 OE, respectively. β-Actin is shown as an input and specificity control. F, Densitometric quantification of MIB1 and BMPR2 protein levels from the input immunoblots shown in E. Signals were normalized to β-Actin and expressed relative to the MIB1 WT (no cDNA, −Dox) control.