Journal: The Journal of Cell Biology
Article Title: ISM1 regulates NODAL signaling and asymmetric organ morphogenesis during development
Figure Lengend Snippet: The inhibitory effects of ISM1, LEFTY1, and CER1 on NODAL signaling. (A) Serum-starved HEK293T-CRIPTO cells treated with 100 ng/ml rNODAL in the presence of mock, LEFTY1, ISM1, or CER1 CM at indicated dilutions. WCL samples were subjected to Western blotting analyses for pSMAD2. Total SMAD2/3 and ÃÂ²-actin were used as internal controls. The protein levels of LEFTY1, ISM1, and CER1 representing their relative concentrations in the CMs were analyzed by Western blotting using Flag antibodies. (B) Quantification of the intensity of pSMAD2 relative to SMAD2/3 in three biological replicates. Data represent mean ÃÂ± SEM. The unpaired, two-tailed t test was used for statistical analyses. Statistically significant P values are indicated. (C) Dual luciferase reporter assay to determine the SMAD2/FOXH1 transcriptional activity in HEK293T-CRIPTO cells treated by 100 ng/ml rNODAL for 24 h, together with CM of mock, FLAG-LEFTY1, FLAG-ISM1, or FLAG-CER1 at the indicated dilutions. Data represent mean ÃÂ± SEM of four independent experiments. P values of statistical analyses using unpaired, two-tailed t test are indicated in each individual experimental group.
Article Snippet: MYC-ACVR2A and CER1-FLAG plasmids were purchased from Sino Biological (MG50613-NM and MG51161-CF, respectively).
Techniques: Western Blot, Two Tailed Test, Luciferase, Reporter Assay, Activity Assay