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7 Pfizer Licensed Compound Library Procedures Unleashed


6 �� 10.4%; pAb-SA3, ?62.7 �� 3.3%; pAb-BD5, ?65.2 �� 17.6%; mAb-BD5, ?58.6 �� 18.1%; and IC50, pAb-UB1, 0.7 �� 0.3??g��mL?1; #links# pAb-SA3, 0.5 �� 0.2??g��mL?1; pAb-BD5, 0.2 �� 0.1??g��mL?1; mAb-BD5, 0.3 �� 0.2??g��mL?1). As previously observed, mAb-RDS6 was ineffective on rodent trkB. Finally, mAb-AC7 was not able to activate rodent TrkB in neurones. Together with the lack of labelling previously observed in Western blots (Figure?1B), these data confirm that mAb-AC7 specifically bind and activate human but not rodent trkB. The pharmacological characteristics (intrinsic activities relative to BDNF, EC50 and IC50) of all antibodies are summarized in Table?2. It should be noted that control experiments performed with respective reconstitution buffer of each antibody (as described by the manufacturer) in the presence or in the absence of BDNF ruled out possible non-specific effects (data not shown). To investigate the molecular mechanism underlying these effects on trkB receptors, binding experiments using iodinated BDNF and KIRA-elisa assays were performed in the native system (i.e. cortical neurones co-expressing native trkB and p75NTR). Consistent with results described previously in neuronal cells, mAb-RDS6 and mAb-AC7 did not alter the BDNF concentration-response curve (Figure?3A,B). Neurone-active partial agonists pAb-UB1 and pAb-SA3 partially #links# decreased the BDNF response in a non-competitive way, as demonstrated by the decrease in maximal response (pAb-UB1, ?71.7 �� 3.7%; pAb-SA3, ?37.7 �� 5.5%) and no significant change in BDNF EC50 (BDNF alone, 117.9 �� 20.5?pM; BDNF + pAb-UB1, 162.6 �� 30.7?pM; BDNF + pAb-SA3, 110.2 �� 55.3?pM). Conversely, antagonist antibodies pAb-BD5 and mAb-BD5 decreased #links# the BDNF response through a competitive mechanism, as shown by the rightward shift of the BDNF curve (BDNF + pAb-BD5, 385.6 �� 12.7?pM, P < 0.01, one-way anova, n= 3; BDNF + mAb-BD5, 1.10 �� 0.17?nM, P < 0.01, one-way anova, n= 3) and no significant change in maximal effect (Figure?3A). All these observations were confirmed by an Eadie�CHofstee plot of the curves (Figure?3B). Binding studies using [125I]-BDNF on both recombinant human and rodent neuronal trkB receptors also revealed distinct binding properties for polyclonal antibodies (Figure?3C). In fact, while pAb-UB1 and pAb-SA3 were able to prevent the binding of [125I]-BDNF to trkB receptors, pAb-BD5 had no effect. Interestingly, pAb-UB1, which was raised against the rodent form, was more efficient in rodent neurones than in TetOn-rhtrkB cells (human, 125.4 �� 23.9??g��mL?1 and rodent, 1.2 �� 0.3??g��mL?1, P < 0.001, t-test, n= 2�C4). Conversely, pAb-SA3, which was originally designed to recognize the human form, was more efficient in TetOn-rhtrkB cells than in neuronal cultures (human, 0.7 �� 0.2??g��mL?1 and rodent, 4.8 �� 0.1??g��mL?1, P < 0.01, t-test, n= 2�C4).