Figure 4.

Immuno-detection of the α₁ skeletal subunit of the DHPR in GLT-α₁–transfected cells. Cells were fixed and incubated with an anti-α₁ subunit of DHPR antibody (see materials and methods) and a rhodamine-conjugated secondary antibody. GLT-α₁–transfected (A and B) cells are shown as both a projected reconstruction (A, Bar = 25 μm) or a series of confocal sections (B, Bar = 20 μm). The pattern of distribution of this subunit is punctate, especially near the surface (in A, small white dots and in B, at levels of 3.5, 4.0, and 4.5 μm) that most likely represent peripheral couplings. Some staining is found diffusely within the cytosol. Untransfected (C, Bar = 30 μm) and mock-transfected GLT cells (GLT-NEO) (D, Bar = 30 μm) were also evaluated for the presence and location of the α₁ subunit of the DHPR. No reactivity above background was observed in either the untransfected or mock-transfected cells (C and D). The brightest optical sections were chosen from each sample. E. Western blots for the anti-α₁ subunit of DHPR (top) in cell homogenates of GLT-α₁–transfected cells, untransfected GLTs, and control NLT cells. The bottom panel shows immunoreactivity for anti–β-tubulin, a marker for protein loading. Note that the amount of protein loaded for NLT cells is one tenth of the amount needed for a similar reactivity for GLT-α₁–transfected cells.