Abstract
To search for caveolar proteins, mice were immunised with rat adipocyte membranes. Hybridoma supernatants were screened for antibodies to proteins on the cytosolic face of caveolae by indirect immunoelectron microscopy of immunogold-labelled adipocyte plasma membrane sheets adsorbed on electron-microscope (EM) grids. One of the hybridoma supernatants (2F11) produced a specific labelling of caveolae which was much more intense than that obtained with caveolin-1 antibodies. In Western blots of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separated proteins in crude membrane fractions from different rat tissues, 2F11 labelled a band corresponding to 60 kDa. The intensity of 2F11 labelling was high in adipose tissue and in other tissues varied in parallel to caveolin- labelling. In blots of plasma membrane (PM) and light-microsomal (LM) fractions from a homogenate of adipocytes, prior insulin stimulation of the adipocytes translocated GLUT-4 from the LM to the PM fraction, but was without effect on the distribution of the 60-kDa protein labelled by 2F11. Digestion with endoproteinase lys-C produced the same pattern of immunoreactive fragments of the protein in the vesicular PM and LM fractions, indicating similar membrane topology of the 2F11-reactive, 60-kDa protein in vesicles of PM and LM fractions.
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