Journal: Folia Microbiologica

Article Title: Microbial transglutaminase and its application in the food industry. A review

doi: 10.1007/s12223-013-0287-x

Figure Lengend Snippet: Strains selected for production of microbial transglutaminase (MTGase)

Article Snippet: The activity of transglutaminase at 1.8–3.4 U/mL was generated by S . mobaraense DSMZ 40847 cultured on a medium with dissolved starch and peptone (Zheng et al. ; Zhu et al. ); this culture spanned 36–60 h. Transglutaminase synthesised by the Streptomyces sp. Strain, cultured for 7 days on a medium with starch, was active at 0.05–0.2 U/mL (Iancu et al. ).

Techniques:

Journal: Scientific Reports

Article Title: C-terminal deletion of NOTCH1 intracellular domain (N1 ICD ) increases its stability but does not amplify and recapitulate N1 ICD -dependent signalling

doi: 10.1038/s41598-017-05119-0

Figure Lengend Snippet: Elevated N1 ICDdC expression levels as compared to N1 ICD do not lead to higher expression of Notch responsive genes. ( a ) U2OS GFP-N1 ICD and GFP-N1 ICDdC cell populations were induced with doxycycline (Dox) for the indicated time period. HES1 and ACTIN expression levels were analysed using specific antibodies. Representative immunoblots are shown. Cropped blots are displayed and full-length blots are included in Supplementary Information. A graphical representation of the mean HES1 expression levels ± SEM of 4 independent experiments is shown where HES1 expression levels were normalized to ACTIN and HES1/ACTIN ratio in uninduced cells was set at 1. ( b ) Uninduced U2OS GFP-N1 ICD and GFP-N1 ICDdC cells were transfected with the Hes1-luciferase and Renilla-luciferase reporter constructs, and cells were left uninduced (without dox) or induced with doxycycline (with dox) for 24 h. The experiment was performed twice in triplicate. The data are expressed as the means ± SEM of Hes1-luciferase activity/Renilla-luciferase activity where the relative activity in uninduced cells (without dox) was set at 1. ***p < 0.001, **p < 0.01 as compared with uninduced cells. ns = not significant. ( c ) Uninduced U2OS GFP-N1 ICD and GFP-N1 ICDdC cells were transfected with the CSL-luciferase and Renilla-luciferase reporter constructs, and cells were left uninduced (without dox) or induced with doxycycline (with dox) for 24 h. The experiment was performed 4 times in triplicate. The data are expressed as the mean ± SEM of CSL-luciferase activity/Renilla-luciferase activity where the relative activity in uninduced cells (without dox) was set at 1. ***p < 0.001 as compared with uninduced cells. ##p < 0.01. ( d ) MIA PaCa-2 cells were transfected with pDEST53 (empty vector), pDEST53-N1 ICD (N1 ICD ) or pDEST53-N1 ICDdC (N1 ICDdC ) together with the Hes1-luciferase or CSL-luciferase and Renilla-luciferase reporter constructs. Luciferase activities were measured the following day. The experiment was performed 3 times in quadruplicate. The data are expressed as the mean ± SEM of Hes1-luciferase or CSL-luciferase activity/Renilla-luciferase activity where the relative activity in empty vector transfected cells was set at 1. *p < 0.05, ***p < 0.001 as compared with empty vector transfected cells. ###p < 0.001. ns = not significant. ( e ) MIA PaCa-2 cells were transfected with pDEST53 (empty vector), pDEST53-N1 ICD (GFP-N1 ICD ) or pDEST53-N1 ICDdC (GFP-N1 ICDdC ). The following day, total cell lysates were analysed for N1 ICD and N1 ICDdC expression using an anti-GFP antibody. An anti-NOTCH1 antibody was also used for detection of endogenous NOTCH1 and GFP-N1 ICD . The asterisk * denotes the expected molecular weight of GFP-N1 ICDdC not detected by the anti-NOTCH1 antibody. Expression levels of MAML1, CSL, HES1 and ACTIN were analysed by immunoblotting using specific antibodies. Cropped blots are displayed and full-length blots are included in Supplementary Information.

Article Snippet: For luciferase assays, cells were transfected with firefly-luciferase reporter construct (CSL-luciferase, gift from Nicholas Gaisano Addgene plasmid #26897 ; Hes1-luciferase , kind gift from Ruth S. Slack, Ottawa), Renilla-luciferase reporter plasmid (pRL-thymidine kinase luciferase) and an expressing vector when indicated (pDEST53, pDEST53-N1 ICD or pDEST53-N1 ICDdC ).

Techniques: Expressing, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Molecular Weight