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. 2013 May 6;110(21):E1923–E1932. doi: 10.1073/pnas.1303916110

Fig. 2.

Fig. 2.

Phenotypes of Lmnb1CS/CS and Lmnb2CS/CS MEFs. (A and B) Metabolic labeling studies showing an absence of lamin B1 and lamin B2 farnesylation in Lmnb1CS/CS and Lmnb2CS/CS MEFs, respectively. Wild-type (Lmnb1+/+ and Lmnb2+/+), Lmnb1CS/CS, and Lmnb2CS/CS MEFs were incubated with the farnesol analog AG, and proteins labeled with AG were detected by Western blotting with an AG-specific monoclonal antibody (31). The lamin B1 in Lmnb1CS/CS MEFs and the lamin B2 in Lmnb2CS/CS MEFs were not labeled by AG. (C and D) Altered electrophoretic mobility of nonfarnesylated versions of lamin B1 and lamin B2. Protein extracts from Lmnb1+/+, Lmnb1CS/CS, Lmnb2+/+, and Lmnb2CS/CS MEFs were size-fractionated on 7% (wt/vol) Tris-Acetate SDS/PAGE gels, and Western blots were performed with antibodies against lamin B1, lamin B2, or lamin C. The nonfarnesylated lamin B1 in Lmnb1CS/CS MEFs and the nonfarnesylated lamin B2 in Lmnb2CS/CS MEFs migrated more slowly than farnesyl–lamin B1 and farnesyl–lamin B2 in wild-type cells. (E) Immunofluorescence microscopy of Lmnb1CS/CS and Lmnb2CS/CS MEFs with antibodies against LAP2β (lamin-associated polypeptide 2; green) and lamin B1 (red) or lamin B2 (red). Images along the z axis were captured and merged (Zen 2010 software; Zeiss). In many Lmnb1CS/CS MEFs, LAP2β and lamin B1 were distributed in a honeycomb fashion. (Scale bar, 20 µm.) (F) Optical sections through a nucleus of a Lmnb1CS/CS MEF along the z axis (Left, top area; Center, middle area; Right, bottom area). Despite nuclear honeycombing, lamin B1 was concentrated at the nuclear rim (Center). (Scale bar, 10 µm.) (G) Frequency of honeycomb nuclear phenotype in primary MEFs. For each cell line, >100 cells were scored by two independent observers in a blinded fashion. From left to right, WT (Lmnb1+/+), n = 3 cell lines; B1CS (Lmnb1CS/CS), n = 5; WT (Lmnb2+/+), n = 3; B2CS (Lmnb2CS/CS), n = 3; WT (Lmnb1+/+), n = 3; HET (Lmnb1+/−), n = 4; and B1KO (Lmnb1−/−), n = 2. The honeycomb nuclear phenotype was more frequent in Lmnb1CS/CS cells than in Lmnb1+/− cells (P < 0.0001). Values represent mean ± SD.