Skip to main content
. 2012 Oct 5;91(4):754–759. doi: 10.1016/j.ajhg.2012.08.024

Figure 4.

Figure 4

Immunoblot and Immuofluorescence Analysis of COS7 Cells Transiently Expressing Wild-Type and Mutant AAGAB and Allele-Specific PCR

(A) COS7 cells were transiently transfected with V5-tagged AAGAB wild-type or mutant (p.Arg124∗ or p.Arg161∗) constructs with the use of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions and were harvested 30 hr after transfection. After lysis of cells in ripa buffer and sonication, lysates were subjected to electrophoresis and immunoblotting. We used monoclonal V5 antibody (R960-25, Invitrogen, 1:4.000) to detect AAGAB proteins. For normalization, we used a monoclonal GAPDH antibody (G8795, Sigma-Aldrich, 1:16.000). The wild-type AAGAB has a size of around 35 kDa, whereas the two truncated proteins have the anticipated sizes of 14 kDa and 18 kDa. However, we observe slightly larger bands, which might be due to the added tag and to glycosylation or phosphorylation of the protein.

(B) Allele-specific PCR was performed with cDNA that was generated from RNA extracted from the blood of affected individuals. We used the same forward primer (5′-ATGATGCTGTGAGATTTTATCCC-3′) for all reactions. The reverse primers used for studying AAGAB expression in individual 54187 were 5′-CACCATTCTTGAGCTTTTTGGCG-3′ for amplifying the wild-type allele and 5′-CACCATTCTTGAGCTTTTTGGCA-3′ for amplifying the mutant c.370C>T allele. The reverse primers used for studying AAGAB expression in individual 60698 were 5′- CATTCAGGGCTTGGACAATGCG-3′ for amplifying the wild-type allele and 5′-CATTCAGGGCTTGGACAATGCA-3′ for amplifying the c.481C>T mutant allele. We readily observed the wild-type allele in both cases, whereas the mutant allele was only present at reduced levels.

(C) Sequencing results of cDNA samples of affected individuals with gene-specific AAGAB primers. We could only detect a very small peak for the c.481C>T allele (arrow on the right), and we could not detect the c.370C>T allele at all (arrow on the left), indicating no presence of the respective mutant transcripts.

(D) Immunofluorescence analysis of transiently transfected HaCaT cells with the above-mentioned V5 antibody. No differences in staining were observed between cells expressing the wild-type (left) or the mutant (center = mutant p.Arg124∗; and right = mutant p.Arg161∗) AAGAB.