FIG. 5.

Truncated Flnc is expressed at low levels in the knockout mice. (A) The knockout is expected to have a truncated protein lacking the last four repeats and the hinge 2 region, but the FLNc unique region against which the FLNc antibodies are made is retained. (B) To determine the amount of truncated protein present, 15 μg of total lysates obtained from limbs of the pups was separated by SDS gel electrophoresis. The Western blot analysis was performed using Flnc-A2 antibodies raised against the unique region. Homozygotes (H) showed a faint band (arrow), whereas the heterozygotes (Het) showed both the wild-type (WT) and truncated alleles. Comparison of the bands in the heterozygotes indicates that the truncated protein is made at very low levels. Western blot analysis with β-actin and sarcomeric α-actinin was performed as a loading control. (C) The amount of Flnc transcript was determined using quantitative RT-PCR. Total RNA was isolated from limb muscles and hearts of homozygous (n = 3 and n = 2), heterozygous (n = 3 and n = 2), and wild-type (n = 1) littermates. The amount of mRNA was first normalized to endogenous Tbp expression in the control and then to the amount of Flnc transcript present in the wild-type animal. The error bars represent the standard errors of the means.