nd grdul increses in BDNF concentrtion elicit distinct signling nd functions in neurons Yunyun Ji,, Yun Lu, Feng Yng, Wnhu Shen, Tin Tze-Tsng Tng,, Linyin Feng, Shumin Dun, nd Bi Lu,.. - Grdul (normlized to Totl ) Grdul (normlized to Totl ) Grdul Supplementry Figure. Kinetics of ctivtion nd signling in response to low concentrtion of BDNF stimultion. Cultures were treted with BDNF in the sme cute or grdul modes descried in Fig., except tht. nm (. ng/ml) BDNF ws used s the finl concentrtion insted of nm (ng/ml). Representtive Western lots () nd quntittive plots () of the time courses of BDNFinduced nd ctivtion in cute nd grdul modes. Nture Neuroscience: doi:./nn.
Grdul -. -... - - GTP GDP ctive Rs Rs ctive Rs Active Rs (normlized to Totl Rs)..... Grdul - - - Rs -. -... - - GTP GDP Grdul ctive Rp Rp Active Rp (normlized to Totl Rp)..... - Grdul ctive Rp Rp - - Supplementry Figure. Kinetics of Rs nd Rp ctivtion in response to cute or grdul BDNF stimultion. Cultures were treted with BDNF in the sme cute or grdul modes descried in Fig.. Active (GTP ound) Rs or Rp ws ffinity-precipitted from cell lystes nd immunolotted with specific ntiodies. Rs () nd Rp () ctivities were quntified y normlizing ctive GTP-Rs nd GTP-Rp to totl Rs nd Rp, respectively. GTPγS or GDP ws dded to unstimulted neuron lystes s positive or negtive controls. Representtive immunolots for Rs-GTP, totl Rs, Rp-GTP, nd totl Rp re shown on top of the quntittive plots. Nture Neuroscience: doi:./nn.
Grdul -. -... - - pplcγ PLCγ pgskβ GSKβ pplcγ PLCγ pgskβ GSKβ pplcγ (normlized to Totl PLCγ) pgskβ (normlized to Totl GSKβ) - Grdul - - - - - Grdul Supplementry Figure. Trnsient or sustined GSK-β nd PLC-γ signling induced y cute or grdul BDNF stimultion. Cultures were treted with BDNF in the sme cute or grdul modes descried in Fig.. Representtive Western lots () nd quntittive plots () of the time courses of BDNF-induced GSK-β nd PLC-γ phosphoryltion in cute nd grdul modes. Nture Neuroscience: doi:./nn.
Grdul -. -... - - (normlized to Totl ) - - - Grdul (normlized to Totl ) - Grdul - - Supplementry Figure. Kinetics of nd ctivtion in response to cute or grdul BDNF stimultion in p NTR-/- culture. Cultures were treted with BDNF in the sme cute or grdul modes descried in Fig.. Representtive Western lots () nd quntittive plots () of the time courses of BDNF-induced nd ctivtion in cute nd grdul modes. Nture Neuroscience: doi:./nn.
Grdul -. -... - - pcreb CREB pcreb pcreb (normlized to Totl CREB)...... Grdul CREB - - - pcreb/dapi (normlized to control)... Grdul min h min Grdul min h Grdul h Supplementry Figure. The durtion of CREB phosphoryltion in response to cute nd grdul BDNF stimultion. () Biochemicl nlysis of CREB ctivtion. Representtive immunolots for pcreb nd totl CREB were presented on top of the quntifiction r grph. N = independent experiments. () Immunostining nlysis of CREB ctivtion. Neurons were fixed nd stined with nti-pcreb ntiody (drk) t the indicted time points under different conditions. Cultures were co-stined with DAPI to revel the loction of neuronl nucleus. Quntifiction dt re presented s the percentge of neurons with nucler pcreb, which ws normlized to control (untreted cells, s ""). Note tht t the " hour" time point, there re more neurons with nucler pcreb under the "grdul" condition s compred to the "cute" condition. Nture Neuroscience: doi:./nn.
- - - - min - - min + + h h h h h h - - - - - - + + + Grdul BDNF nm BDNF nm K nm (normlized to Totl ) (normlized to ) (normlized to Totl ) (normlized to ) Grdul Grdul + K Supplementry Figure. Effects of K on the iologicl response elicited y grdul BDNF dministrtion. Cultures were treted with K ( nm) fter h of grdul BDNF ppliction nd lysed t the indicted time points. Representtive Western lots () nd quntittive plots () re shown. Nture Neuroscience: doi:./nn.
- - - - min - - min + + h h h h h h - - - - - - + + + Grdul BDNF nm BDNF nm U µm (normlized to Totl ) (normlized to ) 9 (normlized to Totl ) (normlized to ) Grdul Grdul + U Supplementry Figure. Effects of U on the iologicl response elicited y grdul BDNF dministrtion. Cultures were treted with U ( µm) fter h of grdul BDNF ppliction nd lysed t the indicted time points. Representtive Western lots () nd quntittive plots () re shown. Nture Neuroscience: doi:./nn.
Initil Slope (%) week old ml/hour ( mice, slices) Initil Slope (%) week old ml/hour ( mice, slices) ng/ml BDNF - ng/ml NGF Time min - nm K Time min Supplementry Figure. Effects of NGF or K on sl synptic trnsmission. () Fst perfusion of NGF ( ng/ml) hs no effect on sl synptic trnsmission ( + _ % t -min). () Incresed synptic trnsmission y fst perfusion of BDNF ws locked y nm K ( _ + % t -min). Nture Neuroscience: doi:./nn.
(kd) M Sline Grdul -. -... - -.. M (kd) (kd) s Surfce min h h min Grdul h h Totl cell lyste for totl (kd) Sline Grdul -. -... - -.. (kd) (kd) s min h Totl h min Grdul h h c (kd) M Grdul -. -..... - - (kd) M d (kd) ml h - ml h - ml h - ml h -. (kd). (kd). (kd). e (kd) ml h - ml h - M. (kd). M Supplementry Figure 9. Protein lots. () Immunolot of the experiment shown on figure. () Complete imges of the experiment shown on figure c. (c) Immunolot of the experiment shown on figure. (d) Immunolot of the experiment shown on figure. (e) Immunolot of the experiment shown on figure d. Uncropped imges of the lots presented on figure, nd cn not e shown since memrnes were cut in two pieces tht were immunolotted individully. M: Moleculr weight mrkers. Nture Neuroscience: doi:./nn. 9