Fig 1: Regulation of JNK, PKC, and ERK1/ERK2 phosphorylation by pharmacological reagents in MSNs in the presence and absence of CD40L reverse signalling. (a–c) Representative western blots of lysates of MSNs of Cd40−/− E14 embryos cultured for 9 days and treated for 20 min with either 1 μg/mL control Fc or 1 μg/mL CD40-Fc in combination with activators (green) and inhibitors (red) of PKC, JNK, and ERK as indicated. The concentrations were the same as those indicated in Figure 2. The western blots were probed with anti-pJNK after treatment with activators and inhibitors of PKC and ERK1/ERK2 (a), anti-pPKC after treatment with activators and inhibitors of ERK1/ERK2 and JNK (b), and anti-pERK1/pERK2 after treatment with activators and inhibitors of PKC and JNK (c). Anti-βIII tubulin was used to normalize western blots of cytosolic fractions and Naphthol blue for the nuclear fractions. (d–f) Quantification of at least three independent western blots. The grey bars show combined treatments in the presence of 1 μg/mL CD40-Fc, and the white bar, the control with 1 μg/mL Fc. The mean ± s.e.m are indicated (*** p < 0.001, ** p < 0.01, and * p < 0.05, one-way ANOVA with multiple Newman–Keuls statistical comparison).
Fig 2: Protein kinase C (PKC), extracellular regulated kinases 1 and 2 (ERK1/2), and c-Jun N-terminal kinase (JNK) phosphorylation after stimulating CD40L reverse signalling. (a,b) Representative western blots of lysates of Cd40−/− E14 striatal medium spiny neuron (MSN) cultures treated for the indicated times with (a) 1 μg/mL CD40-Fc or (b) 1 μg/mL Fc protein as a control. Lysates were prepared from all cultures after a total of 9 days in vitro. The blots were labelled with anti-phopho-PKCThr514 (pPKC), anti-phospho-p44/p42Thr202/Tyr204 MAPK (ERK1/2) (pERK 1/2), anti-phospho-SAPK/JNKThr183/Tyr185 (pJNK). Anti-PKC (PKC), anti-p44/p42 (ERK1/2), and anti-βIII tubulin (βIII tub) were used as loading control for the cytosolic fractions and naphthol blue was used as loading control in the nuclear fraction. (c) Densitometry of at least three independent western blots using βIII tubulin (βIII tub) for normalising pPKC and pERK1/2 and naphthol blue for pJNK (mean ± s.e.m.).
Fig 3: The influence of pharmacological reagents in combination on dendrite growth from hippocampal pyramidal neurons. a–c Scatter charts of total dendrite lengths of hippocampal pyramidal neurons of E18 Cd40−/− embryos cultured for 9 days in vitro and treated 24 h after plating with 1 μg/ml CD40-Fc (grey bars) plus either activators or inhibitors of JNK (a), PKC (b) or ERK1/ERK2 (c) in combination with the activators or/and inhibitors of the other two pathways (the same concentrations were used as in Fig. 3). For comparison, dendrite lengths of neurons in cultures treated with 1 μg/ml control Fc alone (clear bars) are shown. The mean ± s.e.m of at least three independent experiments is shown. The dots represent the data obtained from individual neurons (mean of > 50 neurons per condition). One-way ANOVA with multiple Newman–Keuls statistical comparison, ***p < 0.001, **p < 0.01 and *p < 0.05
Fig 4: CD40L reverse signaling restores the expression of PSD-95, RhoA/B/C, and Cdc42 and resembles the spine morphology in Cd40–/– cultured neurons. (A) Illustrative Western blots of the expression of PSD-95, synaptophysin, RhoA/B/C, Cdc42, and Rac1/2/3, using anti-βIII tubulin and anti-GAPDH as loading controls in Cd40–/– cultured neurons treated with Fc or CD40-Fc 24 h after seeding and cultured during the indicated days. (B) Quantification of the relative expression of these proteins normalized to βIII tubulin from Western blots from at least three independent cultures. Graphs show mean ± s.e.m. One-way ANOVA with multiple Newman-Keuls statistical comparison. ∗∗p < 0.01 and ∗p < 0.05. (C) Immunocytochemical localization of DARPP-32 (green), PSD-95 (red), and merge (yellow) in 18 days cultures of Cd40–/– treated 24 h after seeding with Fc or CD40-Fc. In the DARPP-32 images, ∗ indicates spines in 40 μm fragments. In the merge images, the arrows indicate the co-localization of PSD-95 and DARPP-32 in spines and the dashed arrows indicate co-localization of these proteins in the base of spines. Scale bar 5 μm. Quantification of total number of spines and of the type of spines (branched, mushroom, stubby, thin, and filopodia) per 50 μm dendrite fragment. The means ± s.e.m from 50 μm fragments obtained from at least three independent cultures of Cd40–/– treated with Fc (41, dark gray bars) and CD40-Fc (68, pale gray bars). The dots represent the total number of spines counted. T-test ∗∗p < 0.01 and ∗p < 0.05.
Fig 5: Pull down of Syk in Cd40−/− striatal MSNs after CD40-activated CD40L reverse signalling. (a) Representative western blots of the expression of CD40L, Syk, PKCβ, and PKCγ in Cd40−/− neurons from E14 embryos cultured for 9 days and treated for 30 min with either 1 μg/mL Fc or 1 μg/mL CD40-Fc, before pulled-down Fc fragment (input) and after pulled-down Fc fragment (IP). * = nonspecific band. (b) Quantification of at least three independent western blots of the quantity of CD40L, 40 kDa Syk, 72 kDa Syk, PKCβ, and PKCγ after IP normalizing to the total quantity of those proteins in the input. T-test: ** p < 0.01 and * p < 0.05.
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