Journal: Orphanet Journal of Rare Diseases

Article Title: A hypomorphic BMPR1B mutation causes du Pan acromesomelic dysplasia

doi: 10.1186/s13023-015-0299-5

Figure Lengend Snippet: The Arg31Cys substitution in BMPR1B causes a moderate loss of function. a NIH/3 T3 cells were co-transfected with the empty vector pCS2+ or the indicated HA-tagged variants of Bmpr1b with the luciferase constructs BRE-pLG3ti and pRL-TK and stimulated with 2 nM of human recombinant GDF5. BMPR1B WT strongly induced luciferase activity. The variant BMPR1B Arg31Cys showed nearly no activity without ligand stimulation. It was activated after GDF5 treatment but could not accomplish the level of BMPR1B WT. Arg31His induced signaling, but considerably lower compared to BMPR1B WT. BMPR1B Cys53Arg did not activate SMAD signaling at all. Data were tested for normal distribution (Kolmogorov-Smirnoff normality test) and analyzed using a One-Way-ANOVA with subsequent Bonferroni's Multiple Comparison Test (n = 8; ns not significant, *** p < 0.001). b NIH/3 T3 cells were transfected with the indicated HA-tagged Bmpr1b variants. Using an anti-HA antibody the expression of BMPR1B could by visualized via confocal microscopy. DAPI was used for staining of cell nuclei. All BMPR1B variants were translocated to the cell membrane

Article Snippet: Cells were transfected in growth medium with the control vector pCS2+ or one of the Bmpr1b variants in pCS2+ together with the BRE luciferase reporter construct BRE-pLG3ti [ ] and the Renilla luciferase normalization vector pRL-TK (Promega).

Techniques: Transfection, Plasmid Preparation, Luciferase, Construct, Recombinant, Activity Assay, Variant Assay, Comparison, Expressing, Confocal Microscopy, Staining, Membrane