Isoform I (mdr3) is the major form of P-glycoprotein expressed in mouse brain capillaries. Evidence for cross-reactivity of antibody C219 with an unrelated protein.
P-glycoprotein (P-gp) is expressed in various non-cancerous tissues such as the endothelial cells of the blood-brain barrier. We used several monoclonal antibodies (mAbs) and isoform-specific polyclonal antibodies to establish which P-gp isoforms are expressed in isolated mouse brain capillaries. P-gp class I isoform was detected in capillaries with a Western immunoblotting procedure using a specific antiserum. No immunoreactivity was observed with either class II- or class III-specific antisera. Immunoreactivity was observed with mAb C219. However, this antibody detected two distinct immunoreactive proteins (155 and 190 kDa) in the isolated brain capillaries. These two proteins comigrated as a broad band when the samples were submitted to heat prior to gel electrophoresis. The glycoprotein nature of these two antigens was evaluated by their sensitivity to N-glycanase treatment. Following this treatment, the size of the proteins was reduced from 190 and 155 kDa to 180 and 120 kDa, respectively. Triton X-114 phase-partitioning studies showed that the 190 kDa immunoreactive protein was poorly solubilized by Triton X-114, while the 155 kDa protein was partitioned in the detergent-rich phase. In labelling experiments, only the 155 kDa protein was photolabelled with [125I]iodoarylazidoprazosin. These results show that a 190 kDa protein detected by antibody C219 is an antigen unrelated to the three P-gp isoforms presently known. Cross-reactivity of C219 with an unrelated protein emphasizes the fact that more than one antibody should be used in the assessment of P-gp expression in cell lines and tissues.
Département de Chimie-Biochimie, Université du Québec à Montréal, Canada.
L
L
Jetté
J F
JF
Pouliot
G F
GF
Murphy
R
R
Béliveau
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000941:Antigens / Q000032:analysis; D015153:Blotting, Western; D001921:Brain / Q000098:blood supply*; D002196:Capillaries / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006358:Hot Temperature; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010777:Photochemistry
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D000941:Antigens