The gel pieces were washed three times alternately with 40 mM NH4HCO3 and ethanol. the significance of the lamina far beyond nuclear scaffolding. Thus, lamins are currently considered as key regulators of gene expression by their involvement in signaling, transcription and chromatin organization.20-22 Ongoing characterization of the relationship between physiological functions of nuclear lamins and their contribution to the molecular pathophysiology of laminopathies has greatly benefited from genetically modified mouse models. One of the most versatile models providing insights to various issues of A-type lamin function was provided by Sullivan and colleagues1 who created a mouse line with targeted disruption of the locus. Initially, these mice (gene product in MEFs and tissues Having MEFs1 on hand, Begacestat (GSI-953) we intended to use this cell line as unfavorable control to validate the specificity of new polyclonal antibodies (pAb bs-01) against murine lamin A/C raised in our laboratory. Unexpectedly, though affinity purified against the real immunized epitope, these antibodies showed distinct reactivity with an antigen localized at the nuclear envelope (NE) of MEFs in immunocytochemistry (Fig.?1A-?A). Assuming nonspecific cross-reactivity at first, we were surprised to make the same observation with the established and frequently used A-type lamin antibodies pAb H-110 (Santa Cruz Biotechnology, Santa Cruz, USA) and mAb R2738 since both of them specifically stained the NE of hepatocytes, although signal intensities were slightly reduced compared with wild type controls (compare panels A and E). Consistent with the initial characterization by Sullivan et al.,1 nuclei of MEFs and tissues interacting with pAb bs-01 and pAb H-110 by immunoblotting (Fig.?3). In line with the findings reported previously,1 both antibodies proved the absence of full-length lamins A (70 kDa) and C (60 kDa) in heart, liver and MEFs. However, in these samples both antibodies clearly reacted with a distinct antigen migrating at about 54 kDa. Although both pAb bs-01 and pAb H-110 detected bands of comparable molecular weight (~55 kDa) in wild type controls as well (heart and Begacestat (GSI-953) liver), these antigens migrated slightly higher indicating that they represent proteins Begacestat (GSI-953) different from those found in gene product in MEFs and tissues Implicating apparent inconsistencies with the initial study1 that reported absence of both full-length and truncated lamin A/C proteins in gene performed by Sullivan et al. who replaced exons 8 to a part of 11 by a neomycin resistance cassette, we computationally generated a cDNA encoding a putative gene product consisting of exons 1 to 7 and 12 but lacking exons 8 to 11 (therefore referred to as lamin A?8C11). A corresponding lamin A?8C11 mRNA, which could be produced by alternative splicing in vivo, would exclude the gene knockout construct and would therefore probably give rise to a stable polypeptide. In silico translation (http://expasy.org/tools/pi_tool.html) revealed a predicted mass of a putative lamin A?8C11 protein of 53.5 kDa. This calculated mass would in fact correspond well to the size of the antigen detected in MEFs and tissues using two different lamin A-specific primer sets with binding in exons 1 (5) and 12 (3) each (Fig.?4). Using these primers, amplicons corresponding to full-length Itga6 lamin A (~2 kbp) were readily detectable in wild type controls (heart and liver). As expected from our immunoblots and in line with the data provided by Sullivan et al.1 these amplicons were absent in MEFs and tissues isolated from mice. Instead, in all samples PCR products well matching the size of the putative lamin A?8C11 message (~1.4 kbp) could be amplified. Consistent with the notion that this 54 kDa antigen of mice, the obtained ~1.4 kbp PCR products were cloned and sequenced (Fig.?4). This revealed the expected nucleotide sequence of lamin A?8C11 comprising exons 1C7 and 12 but lacking exons 8C11 of MEFs and tissues at least around the transcriptional level. Open in a separate window Physique?4. RT-PCR reveals the presence of an mRNA encoding the truncated gene product lamin A?8C11 in gene products in are spliced to exon 12. Nucleotides 1C1157 (not depicted) are encoded by exons Begacestat (GSI-953) 1C6 of Nucleotides coding for a CaaX-motif at the C-terminus of the corresponding polypeptide are given in strong. Lamin A?8C11 is a truncated gene product that is present as a stable protein in (i.e., residues 1C460 of lamin A/C) whereas peptides encoded by.