pcDNA3-IR-B-A1×3 (Mut): Mut-GFP was digested with NheI and ApaI restriction enzymes, treated with Klenow and T4 DNA polymerase and then re-ligated to generate Mut.
These results suggest that microarray based sequencing is a useful tool for the detection of mutations in MUT in patients with mut methylmalonic acidemia.
(C) Immunostaining of 5hmC after overexpression of WT TET1 (TET1), TET1 dCXXC (dCXXC), and TET1 CD mut (CD mut).
The plasmids harboring wild type (WT) TET1, TET1 dCXXC (dCXXC), or TET1 CD mut (CD mut) genes, were transfected into Neuro2a cells.
(E) Quantification of the differentiation rates from Neuro2a cells after overexpression of WT TET1 (TET1), TET1 dCXXC (dCXXC), and TET1 CD mut (CD mut).
(C) Western blotting was used to analyze srGAP3 level after overexpression of WT TET1 (TET1), TET1 dCXXC (dCXXC), and TET1 CD mut (CD mut).
(D) Neuro2a cells transfected with control vector (Vector) or vector harboring WT TET1 (TET1), TET1 dCXXC (dCXXC), and TET1 CD mut (CD mut) gene were co-immunostained with Flag and GAP43 antibodies.
(B) Neuro2a cells transfected with control vector (Vector) or vector harboring WT TET1 (TET1), TET1 dCXXC (dCXXC), and TET1 CD mut (CD mut) were lysed and immunoblotted with TET1 antibody.
Mut Tg(INS-Alb-Mut) mice, although completely rescued from neonatal lethality that was displayed by Mut mice, manifested a decreased glomerular filtration rate (GFR), chronic tubulointerstitial nephritis and ultrastructural changes in the proximal tubule mitochondria associated with aberrant tubular function, as demonstrated by single-nephron GFR studies.
The plasmids for 5' UTRs of murine β-actin, L32, L32 mut (non-TOP; C-A mut), S16, S16 mut (non-TOP) and L30 were provided by O. Meyuhas (Hebrew University, Jerusalem, Israel).
In the luciferase activity assay, mutation in E2F1 binding site at −150/−143 bp (Mut-1), −5/+3 bp (Mut-2), +13/+23 bp (Mut-3), and all E2F1 sites (Mut-1.2.3) within the RASSF1A promoter reduced the promoter activity (Fig. 2E).
