Figure 3

Characterization of a newly generated muskelin-specific antibody. (A) Western blot analysis of cell lysate from adult hippocampus. The antibody detects full-length muskelin and a previously reported putative proteolytic fragment. (B) Immunostaining of cultured hippocampal neurons expressing GFP-Muskelin (1–160) (green channel). The muskelin-specific antibody (red channel), originally generated against N-terminal muskelin sequences, recognizes the overexpressed green fluorescent fusion protein, as revealed from the precise colocalization of green and red puncta in distal dendrites (yellow). The red nucleus in the middle of the image represents immunoreactivity of an untransfected neuron close to the analysed cell in a different optical layer. The boxed region is shown as individual channels. Scale bar: 15 μm. (C) Preabsorption control upon antibody clearance with GST-Muskelin 1–280. Neither the preimmuneserum control (left), nor the preabsorption control (right) produce unspecific signals. In contrast, immunostaining with anti-muskelin (middle) reveals distinct immunoreactivity patterns. Scale bar: 15 μm. (D) Analysis of HEK293 cell extracts upon expression of myc-muskelin or empty vector (mock) with either myc- or muskelin-specific antibody. The newly generated antibody against muskelin recognizes a band of the same size, as detected with anti-myc.