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Fig. 3. Production and characterization of polyclonal and monoclonal antibodies specific to the TNGW1 isoform. (A) Rabbit polyclonal anti-rTNR antibodies strongly reacted with rTNR polypeptide coated in ALBIA. Compared with two rabbit anti-GW182 antibodies (5182 and 6642) and the pre-immune rabbit sera, rabbit anti-rTNR sera 6225 and 6226 strongly reacted with rTNR-coated beads. (B) Rabbit anti-rTNR antibodies recognized only TNGW1 but not GW182 in western blots. HeLa cells transfected with EGFP-tagged TNGW1, GW182, rTNR and EGFP were harvested 24 hours after transfection. These cell lysates, together with recombinant 6xHis-tagged rTNR, were analyzed by western blotting using rabbit anti-rTNR sera 6225 and 6226. The membranes shown in columns a and c were first blotted with rabbit anti-rTNR antibodies, stripped and then re-blotted with anti-EGFP antibodies to confirm the recombinant proteins were expressed (b and d, respectively). (C) Reactivity of mouse anti-rTNR mAb with rTNR polypeptide by using ALBIA. Three mouse mAbs (2E11, 5C8 and 2F11) showed a high number of MFUs to rTNR, whereas control mAb anti-GW182 4B6, anti-GW2-25 and culture medium showed little or no reactivity. Note that mAbs 2E11 and 5C8 showed an 
20-fold higher number of MFUs than 2F11. (D) Peptide array mapping of antibody reactivity to the TNR region. Two duplicate peptide array membranes containing 59 sequential 15-amino-acid peptides, each with 5-amino-acid offset spanning the TNR region were synthesized and used to examine antibody specificity. Membrane number 1 (#1, left) was probed sequentially using anti-EEA1control mAb, anti-rTNR mAbs 2E11 and 2F11, and rabbit anti-rTNR 6225. Membrane number 2 (#2, right) was probed sequentially using anti-GW182 mAb 4B6, anti-rTNR mAb 5C8, rabbit anti-rTNR 6226 and human serum 18033. Boxed areas show the peptide strings that reside in TNR Q-repeat domains (as indicated by underlined residues in Fig. 1C). All rabbit and human sera showed reactivity with multiple peptides. The three mouse anti-rTNR mAbs recognized a relatively narrow region outside of the TNR Q-repeat domain.
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