Figure 3

BMPRII-kinase activity is dispensable but BMPRI enhances the p55γ interaction with BMPRII. (A) Co-immunoprecipitation of HA-tagged BMPRII-LF or kinase-dead BMPRII-K230R-HA with flag-tagged p55γ in the presence or absence of BMPRIb-HA from transiently transfected HEK293T cells. Left panel depicts quantification of co-immunoprecipitated BMPRII relative to the amount of flag-tag mediated precipitation of p55γ. Upper panel shows input controls for BMPRII-HA, BMPRIb-HA and p55γ-flag. Lower panel depicts co-immunoprecipitation of BMPRII-HA with blots taken with long (upper lanes) and short (lower lanes) exposure times. White arrowheads indicate the migration heights of BMPRII, BMPRIb and p55γ. Dotted lines indicate deletion of non-relevant lanes from the same blot. (B) Activation of PI3K signalling by transient expression of BMPRII and BMPRII-K230R. Upper panel shows the effect of increasing amounts of BMPRII-LF or BMPRII-K230R transfected cells against β-galactosidase (β-gal) on the activation of phospho-Akt Thr308 in HEK293T cells. Experiments were carried out in the presence of 10 nM BMP2, which was added 60 minutes prior to cell lysis. Note the dominant negative effect of BMPRII-LF-K230R on pSmad1/5/8 but not phospho-Akt Thr308. Lower panel shows quantification of phospho-Akt Thr308 intensities relative to GAPDH. Error bars represent standard deviation from three independent experiments. P-values from one-way analysis of variance with post-hoc Bonferroni-test are indicated. (See also Additional file 2: Figure S2.)