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Functional Requirement for Class I MHC in CNS Development and Plasticity
Gene S. Huh, Lisa M. Boulanger, Hongping Du, Patricio A. Riquelme, Tilmann M. Brotz, and Carla J. Shatz
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Supplementary Material
Class I MHC molecules are positioned to act at synapses.
Immunoblot of synaptosomal preparations from adult rat brain (40
m g/lane) was probed with the anti-rat MHC I monoclonal antibody OX18 (upper panel). In liver (L) and brain homogenate (H), OX18 reacts with multiple species (in the 44- to 55-kD range); the preparation of synaptosomes (SS) enriches for the 44-kD species, suggesting that a subset of class I MHC molecules in the brain are associated with synapses. (Lower panel) the same blot was reprobed with a monoclonal antibody against the synaptic marker synaptotagmin (Syt).
Methods
Adult rat brain synaptosome preparations were prepared essentially as described [A. Nagy, A. V. Delgado-Escueta, J. Neurochem. 43, 1114 (1984)]. Brain homogenate or synaptosomes were separated by 10% SDS-PAGE, transferred to 0.2 mm nitrocellulose (Schleicher & Schuell, Keene, NH), and probed with either OX18 (2 mg/ml, Serotec, Raleigh, NC) or monoclonal antibodies against synaptotagmin (0.5 mg/ml, PharMingen/Transduction Laboratories, San Diego, CA) as described [R. A. Corriveau, G. S. Huh, C. J. Shatz, Neuron 21, 505 (1998)], except that incubations of the primary antibodies were carried out in TBS/0.1% Tween 20/10% Superblock (Pierce, Rockford, IL).
Supplemental Figure 1.
