Journal: Biochemical Journal

Article Title: Activin E is a transforming growth factor β ligand that signals specifically through activin receptor-like kinase 7

doi: 10.1042/BCJ20230404

Figure Lengend Snippet: ( A–C ) Luciferase reporter assay response to recombinant mature ActA, ActC, or GDF11 (5 nM) along with conditioned media of ActE Construct I, Construct II, or empty vector (EV) in (CAGA 12 )-luciferase HEK293T293 cells transfected with SB-431542 resistant ALK4-ST ( A ), ALK5-ST ( B ), or ALK7-ST ( C ) type I receptors. Media was concentrated 20× and diluted two-fold for the titration (2.5×, 5×, 10×, 20×). Data normalized to transfected cells with no ligand treatment. ( D ) Effects of the ALK7 neutralizing antibody (nAb) on recombinant ActA or ActE conditioned media samples on (CAGA 12 )-luciferase promoter activity in cells expressing the designated SB-431542 resistant type I receptor. (ns = not significant) (**** P < 0.0001) (**** P < 0.0001) ( E ). Western blot analysis under reducing and non-reducing conditions of elution samples from the purification of flag-tagged mature ActE from construct 2 using flag affinity resin. ( F ) Luciferase reporter activity of pooled fractions from ( E ) in (CAGA 12 )-luciferase HEK293T cells transfected with the ALK7-ST. Data normalized to untreated control cells. In ( A–D, and F ) each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. In ( A–D, and F ), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Article Snippet: Conditioned media produced with a construct containing GDF8 prodomain and GDF11 mature (8Pro/11Mature) was provided by Elevian.

Techniques: Luciferase, Reporter Assay, Recombinant, Construct, Plasmid Preparation, Transfection, Titration, Activity Assay, Expressing, Western Blot, Purification, Control

Journal: Biochemical Journal

Article Title: Activin E is a transforming growth factor β ligand that signals specifically through activin receptor-like kinase 7

doi: 10.1042/BCJ20230404

Figure Lengend Snippet: ( A ) (CAGA 12 )-luciferase HEK293T cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with recombinant GDF11 (0.62 nM), ActE conditioned media (20×), EV conditioned media (20×), and ActC (0.62 nM) in the presence or absence of increasing amounts of FS288 (12.5 and 25 nM) (**** P < 0.0001) (* P = 0.0105) (*** P = 0.0004) ( P = 0.2746) ( P = 0.8552) (ns = not significant). ( B ) (CAGA 12 )-luciferase HEK293T cells transfected with ALK4-ST or ALK7-ST receptor constructs and treated with recombinant GDF11 (0.62 nM), ActE conditioned media (20×), EV conditioned media (20×), and ActC (0.62 nM) in the presence or absence of increasing amounts of WFIKKN2 (12.5 and 25 nM) (**** P < 0.0001) ( P = 0.3687) ( P = 0.1983) (ns = not significant). Each point represents a technical replicate within triplicate experiments with bars displaying the mean ± SD. Data are normalized to untreated control cells. In ( A and B ), cells were treated with 10 μM SB-431542 to inhibit signaling activity of endogenous receptors.

Article Snippet: Conditioned media produced with a construct containing GDF8 prodomain and GDF11 mature (8Pro/11Mature) was provided by Elevian.

Techniques: Luciferase, Transfection, Construct, Recombinant, Control, Activity Assay