Overlapping expression of Bex1 and p75^NTR mRNAs in developing mesenchyme and vascular and nervous tissues. (A) RT–PCR analysis of Bex1, Bex2 and Bex3 mRNA expression in cultures of PC12 cells, Schwann cells, E17.5 hippocampus and E14.5 dorsal root ganglia (DRG). RT, reverse transcriptase. (B) RT–PCR analysis of Bex1, Bex2 and Bex3 mRNA expression in P1 cerebral cortex, hippocampus and olfactory bulb. (C) Bex1 mRNA expression analyzed by in situ hybridization in sagittal (a–c) and transverse (d, e) sections of E13.5 rat embryos, and in coronal sections of E19.5 rat brain (f–h). me, mesenchyme; he, heart; ao, aorta; sc, spinal cord; mn, motorneurons; drg, dorsal root ganglia; symp, sympathetic ganglia; ob, olfactory bulb; st, striatum; cp, cortical plate; sp, subplate; lv, lateral ventricle; hc hippocampus; th, thalamus.

Dynamic regulation of Bex1 protein levels during the cell cycle and upon neuronal differentiation. (A) PC12 cell cultures were serum-starved for 24 h and then treated with NGF plus 1.5% serum for up to 6 days. Expression of endogenous Bex1 was analyzed by IB. Re-probing for actin was used as loading control. Fold change relative to actin is indicated. Similar results were obtained in three independent experiments. (B) PC12 cell cultures were serum-starved for 24 h and then re-exposed to serum for up to 3 days. Expression of endogenous Bex1 was analyzed by IB. Fold change relative to actin is indicated. Similar results were obtained in three independent experiments. (C) RT–PCR analysis of Bex1 mRNA levels in exponentially growing and serum-starved PC12 cells. RT–PCR for EF-1α was used as loading control. (D) Exponentially growing cultures of PC12 cells were treated with cycloheximide for the indicated periods of time and subsequently analyzed for endogenous expression of Bex1 by IB. (E) Endogenous Bex1 was analyzed by IB in PC12 cells after 1 h serum-starving in the presence or absence of PS I. Levels of Bex1 in exponentially growing cells (15% serum) are shown as control. Reprobing for tubulin was used as loading control.

Deregulated Bex1 expression prevents growth arrest and neuronal differentiation of PC12 cells. (A) The percentage of cells in S phase was determined by flow cytometry analysis of exponentially growing and serum-starved cultures of parental PC12 and clones C4 and E2 overexpressing Bex1. Results shown are the mean±s.e.m. of three independent experiments, each performed in triplicate. ^*P<0.01 versus 15% serum. (B) The percentage of BrdU-positive cells was determined by immunofluorescence analysis of BrdU incorporation in exponentially growing (15% serum), serum-starved and NGF-treated cultures of parental PC12 and clone C4 cells. Counterstaining of nuclei was done with DAPI. Results are shown as average±s.e.m. of three independent experiments each performed in triplicate. ^*P<0.01. (C) The percentage of differentiated cells (i.e. bearing neurites longer than two-cell body diameters) was quantified in parental PC12 and clones C4 and E2 5 days after NGF treatment. Results are shown as average±s.e.m. of three independent experiments each performed in triplicate. ^*P<0.01 versus parental PC12 cells (wt). Representative micrographs of NGF-treated cultures are shown. (D) Percentage of dead cells in parental PC12 cells and clones C4 and E2 after 48 h serum starvation in the presence or absence of NGF. Results are shown as average±s.e.m. of three independent experiments each performed in triplicate.

Bex1 knockdown potentiates NF-κB activity and accelerates PC12 cell differentiation in response to NGF. (A) Bex1 and Bex2 mRNA levels were analyzed by RT–PCR in PC12 cells transfected with a Bex1 siRNA or a control construct. EF1α is shown as loading control. Fold changes relative to control are indicated. (B) Bex1 protein levels were analyzed by IB in PC12 cells transfected with Bex1 siRNA or a control construct together with Flag-Bex1. α-Tubulin is shown as loading control. Fold change relative to control is indicated. (C) The effects of Bex1 knockdown on NF-κB activity were tested in PC12 cells that also received small amounts of ectopic RIP2 (which in our hands enhances NF-κB responses to NGF without altering basal levels), before (medium) or after a 4 h treatment with NGF. Results are expressed as average±s.e.m. of three experiments performed in quadruplicate. ^*P<0.05 versus control. (D) Neuronal differentiation was assessed in synchronized in S phase, PC12 cells transfected with Bex1 siRNA or a control construct as the percentage of cells displaying neurites longer than one or two cell diameters, as indicated (see Supplementary data for details). Results are shown as average±s.d. of a representative experiment performed in triplicate. ^*P<0.05 versus control. Similar results were obtained in two additional experiments.

Identification of Bex1 as a novel intracellular interactor of p75^NTR. (A) Alignment of primary amino-acid sequences of rat Bex1 and mouse Bex1, Bex2 and Bex3 performed with ClustalX (Jeanmougin et al, 1998). The region of rat Bex1 identified by T7 phage display is indicated between arrows. The p75^NTR-binding site in Bex3/NADE is underlined (Mukai et al, 2000). NLS and Ser¹⁰⁵ are indicated. (B) Interaction between HA-tagged Bex1 (HA-Bex1) and p75^NTR overexpressed in 293 cells analyzed by IP with anti-HA antibodies and Western IB with antibodies against the intracellular domain of p75^NTR (p75^ICD). In the lower panel, transfected cells were treated for 12 h with NGF prior to lysis and IP as indicated. (C) IB of cell lysates (50 μg protein) from parental PC12 cells and clones C4 and E2 overexpressing Flag-Bex1 using anti-Flag (left) or anti-Bex1 antibodies. (D) Interaction between endogenous p75^NTR and Flag-Bex1 in clone E2 in the presence or absence of NGF analyzed by IP with control antibodies or antibodies against the extracellular domain of p75^NTR (p75^ECD), followed by IB with anti-Flag antibodies. (E) Interaction between endogenous Bex1 and p75^NTR in parental PC12 cells analyzed by IP with control or anti-p75^ECD antibodies, followed by IB with anti-Bex1 antibodies.

Dynamic nucleocytoplasmic trafficking of Bex1 in response to NGF. (A) 293 cells were transfected with GFP-Bex1, GFP-Bex1^mutNLS or GFP-Bex1 and p75^NTR, and treated with NGF for 2 h as indicated. (B) Subcellular localization of GFP-Bex1 in transiently transfected PC12 cells—before or after 90 min with NGF—was assessed by fractionation and IB with GFP antibodies. CREB and RhoA were used as markers for nuclear and cytoplasmic compartments, respectively. Percentage in each fraction is indicated. A micrograph of cells expressing GFP-Bex1 grown under control conditions is shown to the right. (C) Subcellular localization of Flag-Bex1 in stable transfected PC12 cells (clone E2). (D) Subcellular localization of endogenous Bex1 in naïve PC12 cells. (E) GFP-Bex1 was transiently transfected in parental or mR-1 PC12 cells and the percentage of cells displaying GFP-Bex1 in a predominant cytoplasmic localization following NGF treatment during the indicated times was quantified. A minimum of 900 cells were counted per condition. Results are average of three independent experiments±s.e.m. (F) Translocation of GFP-Bex1 after NGF or BDNF treatment in PC12 cells. ^*P<0.05 versus control. (G) GFP-Bex1 was transiently transfected in Schwann cells and photographed at different times after NGF or control treatments. Arrowheads indicate cells showing cytoplasmic translocation of Bex1 in response to NGF treatment. (H) The number of Schwann cells showing translocation of Bex1 from the nucleus to the cytoplasm after the indicated treatments and times was quantified. Results are percentage of cells showing Bex1 translocation at each time point. In all, 70 cells were observed in each case.

Phosphorylation of Bex1 in Ser¹⁰⁵ by the serine–threonine kinase AKT. (A) Consensus sites for phosphorylation in a subset of known AKT substrates shown together with the corresponding site in the Bex1 sequence. (B) Analysis of ³²P incorporation in HEK293 T cells that had been metabolically labeled with ³²P-orthophosphate after transfection with Flag-tagged versions of wild-type Bex1 or a mutant Bex1 with a Ser¹⁰⁵ to Ala substitution (Flag-Bex1^S105A). An autoradiogram of anti-Flag immunoprecipitates and its corresponding control IB with anti-Flag antibodies is shown. The panel to the right shows the same experiment performed in cells treated with the proteasomal inhibitor PS I, showing that the reduced phosphorylation of the S105A mutant was not due to its otherwise low level of expression. (C) Bex1 phosphorylation analyzed by metabolic labeling of transfected HEK293 T cells following 2 h treatment with the PI3K inhibitor LY294002 or after 24 h serum withdrawal. (D) Kinase assay of bacterially produced GST-tagged Bex1 constructs incubated with lysates of HEK293 T cells transiently transfected with wild-type or kinase-dead versions of AKT. An autoradiogram of GST pull-downs and its corresponding control IB with anti-GST antibodies is shown. GSK3β was used as positive control (lower panels). Arrows denote phosphorylated products. (E) Analysis of AKT activation and Bex1 phosphorylation in PC12 cells treated with NGF. Immunoblots of phospho-AKT, total AKT, phospho-AKT substrates and Bex1 are shown. Phospho-AKT and phospho-Bex1 are indicated. (F) Steady-state levels of Flag-tagged wild type and S105A Bex1 in HEK293 T cells in the presence and absence of the proteasomal inhibitor PS I analyzed by IB with anti-Flag antibodies. Lysates of parental and clone C4 PC12 cells were used as negative and positive controls, respectively.

Bex1 modulates proximal signaling by p75^NTR but not TrkA. (A) NGF-mediated phosphorylation of ERK and AKT was determined in parental PC12 cells and clones overexpressing Bex1 by IB of cell lysates using phospho-specific antibodies. Reprobing of filters with total AKT was used as loading control. (B) NF-κB activity was assessed using a firefly luciferase reporter gene in parental PC12 cells and clones overexpressing Bex1 before or after a 4 h treatment with NGF. Luciferase activity was normalized to that of a control construct carrying a Renilla luciferase gene. Results are expressed as average±s.e.m. of three independent experiments each performed in quadruplicate. ^*P<0.05 versus control. (C) HEK293 T cells were cotransfected with p75^NTR, myc-tagged RIP2 and increasing amounts of Bex1. After a 4 h treatment with NGF, RIP2 was immunoprecipitated from cell lysates with anti-myc antibodies and filters were probed with anti-p75^NTR antibodies. Aliquots of total cell lysates were directly probed with anti-myc and anti-p75^NTR antibodies as control. (D) RIP2 was transiently transfected in parental PC12 cells and clone E2 together with a GFP expression plasmid and then stimulated with NGF. After 2 days, the fraction of differentiated cells was counted in each condition. Results were normalized to the extent of cell differentiation observed in control parental cells (i.e. 25–32% of transfected cells), and are expressed as average±s.e.m. of three independent experiments each performed in triplicate. ^*P<0.05 versus control.

Bex1 inhibits neuronal differentiation of precursors from the brain SVZ. (A) Bex1, Bex2 and Bex3 mRNA levels were analyzed by RT–PCR in floating neurospheres derived from the postnatal SVZ (proliferating) or differentiating neural precursors after 5 days of mitogen withdrawal. EF1α is shown as loading control. (B) Neural precursors transfected with GFP (control) or GFP-Bex1 (Bex1) were allowed to differentiate after mitogen withdrawal. Micrographs show GFP fluorescence, βIII-tubulin immunocytochemistry (in red) and their overlay (merged). In the examples shown, double-positive cells (yellow) are observed in control, but not Bex1-transfected cells. (C) Quantification of neuronal differentiation of SVZ precursors as the percentage of βIII-tubulin-positive cells among the GFP-expressing cell population. Under control conditions (set here to 100%) 20–30% of the cells expressed βIII-tubulin and extended neurites. Average results of three independent experiments are shown ±s.d. ^*P<0.05 versus control.