Article Title: The role of estrogen receptors in rat Sertoli cells at different stages of development
Figure Lengend Snippet: Effects of proteins kinases inhibitors on the expression of non-phosphorylated CTNNB induced by DPN in the Sertoli cells from 20-day-old rats . Sertoli cells from 20-day-old rats were incubated in the absence (C, control) and presence of DPN (10 nM) for 24 h. The cells were also untreated or pretreated with PP2, an inhibitor of the SRC family of protein tyrosine kinases; U0126, an inhibitor of MEK1/2; Wortmannin, an inhibitor of PI3K and MK-2206, an inhibitor of AKT for 30 min. Afterward, the cells were stimulated with DPN (10 nM) 24h. Immunostaining for CTNNB (green) was detected using an antibody specific for non-phosphorylated CTNNB and Alexa Fluor 488-labeled secondary antibody. Nuclei were stained with DAPI (blue). Negative control was performed using normal rabbit serum at the same dilution of antibody. Scale bar as indicated. The data shown are representative of three independent experiments. Note: Control, C, and DPN are the same results of the Figure 5 . All treatments were performed on the same day (Figures 5 and 6 ).
Article Snippet: The cells were also untreated or pretreated with ESR2-selective antagonist PHTPP (4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo(1,5-a)pyrimidin-3-yl)phenol; 10 nM; Tocris Bioscience), the selective inhibitor of the SRC family of protein tyrosine kinases PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine; 5 nM; Calbiochem, Darmstadt, Germany), MEK1/2 inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2- aminophenylthio] butadiene; 20 μM; Cell Signaling Technology), PI3K inhibitor Wortmannin (11-(acetyloxy)-1S,6bR,7,8,9aS,10,11R,11bR-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5-h]-2- benzopyran-3,6,9-trione; 100 nM; Sigma Chemical Co.) or AKT inhibitor MK-2206 (8-(4-(1-aminocyclobutyl)phenyl)-9-phenyl-[1,2,4]triazolo[3,4-f][1,6]naphthyridin-3(2H)-one; 200 nM; Selleckchem, Houston, TX, USA) for 30 min.
Techniques: Expressing, Incubation, Immunostaining, Labeling, Staining, Negative Control