To search for caveolar proteins, mice were immunised with rat adipocyte mem
branes. Hybridoma supernatants were screened for antibodies to proteins on
the cytosolic face of caveolae by indirect immunoelectron microscopy of imm
unogold-labelled adipocyte plasma membrane sheets adsorbed on electron-n-mi
croscope (EM) grids. One of the hybridoma supernatants (2F11) produced a sp
ecific labelling of caveolae which was much more intense than that obtained
with caveolin-1 antibodies. In Western blots of sodium dodecyl sulphate po
lyacrylamide gel electrophoresis (SDS-PAGE) separated proteins in crude mem
brane fractions from different rat tissues, 2F11 labelled a band correspond
ing to 60 kDa. The intensity of 2F11 labelling was high in adipose tissue a
nd in other tissues varied in parallel to caveolin-1 labelling. In blots of
plasma membrane (PM) and light-microsomal (LM) fractions from a homogenate
of adipocytes, prior insulin stimulation of the adipocytes translocated GL
UT-4 from the LM to the PM fraction, but was without effect on the distribu
tion 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.