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Fig. 1. Specificity of guinea pig antibodies for melanosomal proteins. (A) Analysis of antibody reactivities by immunopurification, immunodepletion and western blot analysis of melanocyte extracts. Tyr, Tyrp1, Dct or Silver proteins were immunopurified (left) from an extract of melan-a melanocytes using antibodies as noted at the bottom and then were analyzed by western blot using antibodies as noted on the left. Subscripts of `GP' or `R' refer to antisera generated in guinea pigs or rabbits, respectively; NS, normal serum. An extract of melan-a melanocytes was immunodepleted with antibody and protein-G-Sepharose (middle). Reactivity could be blocked by pre-incubation with the corresponding antigenic peptide (data not shown). Protein extracts from melan-a, melan-b or melan-c melanocytes were separated by SDS-PAGE (right). (B) Intracellular distribution of Tyr and Tyrp1 detected immunohistochemically. Melan-a melanocytes were fixed in 4% paraformaldehyde and stained for Tyr and Tyrp1. Tyrp1-positive (red) vesicles that colocalized with Tyr (green) are seen as yellow in the merged image. (Note that a relatively small fraction of vesicles is yellow, which may reflect cell variability in gene expression or antigen masking in melanized melanosomes.) Bar, 10 µm.
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