Polyclonal antibodies against hNup133 and hNup107 were obtained by injecting recombinant GST-hNup133, 6His-hNup107-N, or 6His-hNup107-C into rabbits (Agrobio). The anti-hNup133 serum was depleted of GST antibodies and affinity purified using GST-hNup133 immobilized on an NHS-activated column. The anti–hNup107-N and anti–hNup107-C antibodies were affinity purified against recombinant GST-hNup107-N and GST-hNup107-C, respectively. Anti–lamin B (Guilly et al., 1987 (link)) and autoimmune CREST serum were obtained from J.C. Courvalin (Institut J. Monad, Paris, France), guinea pig anti-p62 (Cordes et al., 1991 (link)) was a gift from G. Krohne (Biocenter of the University, Würzburg, Germany); SA1 monoclonal antibody against Nup153 (Bodoor et al., 1999 (link)) was provided by Ricardo Bastos (University of Barcelona, Barcelona, Spain); monoclonal anti-p150Glued was from Transduction Laboratories. Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.
For immunofluorescence, cells were either fixed for 20 min in 3% fresh paraformaldehyde and permeabilized with 0.5% Triton X-100 or fixed for 5 min in methanol at −20°C. Ultracryomicrotomy and immunogold labeling of HeLa cells fixed with 4% paraformaldehyde was performed as described (Raposo et al., 1997 ).
For immunofluorescence, cells were either fixed for 20 min in 3% fresh paraformaldehyde and permeabilized with 0.5% Triton X-100 or fixed for 5 min in methanol at −20°C. Ultracryomicrotomy and immunogold labeling of HeLa cells fixed with 4% paraformaldehyde was performed as described (Raposo et al., 1997 ).
