Fig. 4

ACVR2A ^V173I increases ACVR1^R206H?induced BMP signaling in Tg(R206Ha) zebrafish but does not alter signaling in ACVR1 ^R206H HEK 293T cells, while ACVR2A knockdown decreases activin A?induced signaling in ACVR1 ^R206H HEK 293T cells. (A) pSMAD 1/5/9 levels normalized to GAPDH in ACVR1 ^R206H HEK 293T cells after transient transfection with siRNA targeting ACVR2A and stimulation with either BMP4, activin A (AA), or nonstimulated. N ? 5 biological replicates for each condition. Values of p are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (B) Western blot showing pSMAD 1/5/9 levels normalized to GAPDH loading control in ACVR1 ^R206H HEK 293T cells after transient transfection with siRNA targeting ACVR2A and stimulation with either BMP4 or AA. (? = duplicate NT/BMP4 sample repeated across all 3 membranes for normalization). Horizontal boxes denote Western blots that were run on the same membrane but cut between 50 kD and 37 kD prior to separate pSMAD 1/5/9 and GAPDH antibody probing. Values of p are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (C) pSMAD 1/5/9 levels normalized to GAPDH and ACVR2A?DYK protein tag in ACVR1 ^R206H HEK 293T cells after transient transfection with ACVR2A ^WT or ACVR2A ^V173I plasmids. Values of p are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (D) Representative Western blot demonstrating pSMAD 1/5/9 levels normalized to GAPDH and ACVR2A?DYK protein tag in HEK 293T ACVR1 ^R206H cells after transient transfection with ACVR2A ^WT or ACVR2A ^V173I plasmids (representative of n = 3 biological replicates). Values of p are shown numerically with p < 0.05 denoting significance using unpaired, parametric t tests with Welch's correction. (E) (Columns 1?4) Percent ventralized embryos after injection of ACVR2A ^WT (90 ng/?L) or ACVR2A ^V173I (90 ng/?L) mRNA into Tg(ACVR1?R206Ha). Kruskal?Wallis test was performed, statistical significance observed between columns 2 and 4 (p < 0.0001), and columns 3 and 4 (p = 0.0013) (Columns 1?4, n = 46, 56, 27, and 60 embryos, respectively). Parental genotypes are indicated above each column. (F) Uninjected ventralized (V5, V4.5, V4) heat?shocked Tg(ACVR1?R206Ha) embryos. ACVR2A ^WT/V173I mRNAs were injected into separate clutches, and uninjected controls were scored from each clutch. Transgenic?positive and transgenic?negative embryos were sorted via mCherry fluorescence.