anti erbb4 agarose conjugated beads  (Santa Cruz Biotechnology)

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 1. Activated E4ICD Interacts with TAB2 in Yeast and Mammalian Cells (A) E4ICD becomes phosphorylated in yeast. Yeast expressing wild-type (WT) and kinase-dead (KD) LexA-E4ICD fusion proteins were lysed and immunoblotted with ErbB4 or phosphotyrosine (P-Y) antibodies, showing that both proteins are expressed (top panel) but only the WT is tyrosine phosphorylated (bottom panel). (B) Schematic diagram of TAB2. Regions of TAB2 included in two clones identified in the screen are indicated. zf-Ran BD: zinc finger Ran-binding domain. (C) Tyrosine kinase activity of E4ICD is necessary for interaction with TAB2 in yeast. WT or KD LexA-E4ICD was coexpressed in yeast with full-length TAB2 or PDZ domains 1 and 2 of PSD-95 (Huang et al., 2000) as fusion proteins with the B42 activation domain. TAB2 only interacts with phosphor- ylated E4ICD (blue color), whereas PSD-95 interacts with both baits. (D) ErbB4/TAB2 interaction in mammalian cells depends on receptor activation. Full-length ErbB4 WT or KD was transfected into HEK293 cells to- gether with full-length FLAG-TAB2. Cells were left untreated or were treated with NRG1, lysed, and immunoprecipitated with ErbB4 antibody followed by immunoblotting with FLAG antibody. Whole-cell lysates were also immunoblotted with ErbB4, phosphotyrosine (P-Y), and FLAG antibodies to characterize the input.

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Expressing, Clone Assay, Binding Assay, Activity Assay, Activation Assay, Transfection, Immunoprecipitation, Western Blot

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 2. Activated ErbB4 Induces TAB2 Nuclear Translocation and Interacts with N-CoR (A) Activation of presenilin-sensitive isoform ErbB4 JMa induces nuclear translocation of TAB2. Whereas TAB2 immunoreactivity is excluded from the nucleus in unstimulated cells expressing ErbB4 JMa, treatment with NRG1 for 2 hr results in TAB2 nuclear localization. (B) NRG1-induced TAB2 nuclear translocation depends on ErbB4 activation and cleavage. Whereas treatment with NRG1 for 2 hr induces a dramatic increase in TAB2 nuclear localization in cells expressing ErbB4 JMa, this translocation does not occur if cells are exposed to the presenilin inhibitor DAPT 30 min before NRG1 stimulation or if cells express the cleavage-resistant isoform ErbB4 JMb or ErbB4 JMaKD (*p < 0.01). In this and all other figures, error bars represent the standard error of the mean (SEM). (C) ErbB4 JMa-induced TAB2 nuclear translocation represses transcription. Cells expressing ErbB4 JMa or JMb were cotransfected with a GAL4- TAB2 fusion plasmid, a UAS-TK-luciferase reporter, and a TK-renilla reporter. Twenty-four hours later, cells were treated with DAPT or vehicle, followed by stimulation with NRG1. Cells were lysed 24 hr later, and luciferase activity was measured (*p < 0.05). (D) Activated ErbB4 interacts with N-CoR. Cells were transfected with WT or KD ErbB4, treated with NRG1 for 30 min, lysed, and subjected to immunoprecipitation with an ErbB4 antibody followed by immunoblotting with N-CoR. Whole-cell lysates were immunoblotted with ErbB4 and phosphotyrosine antibodies. (E) TAB2 knockdown blocks ErbB4/N-CoR interaction. Cells transduced with control or TAB2 RNAi-expressing lentiviruses were treated and processed as in (D). (F) TAB2 C terminus is necessary for ErbB4/TAB2 interaction. ErbB4 was transfected together with FLAG-TAB2 (full-length or DC truncated protein). Cells were treated and processed as in (D). (G) TAB2 N terminus interacts with N-CoR. Cells were transfected with FLAG-TAB2 (full-length or DC or DN truncated proteins). After lysis and immunoprecipitation with FLAG antibody, samples were immunoblotted with N-CoR antibody. Whole-cell lysates were also immunoblotted with FLAG antibody to characterize the input. FLAG-TAB2, 88 kDa; FLAG-TAB2DC, 80 kDa; FLAG-TAB2DN, 18 kDa.

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Translocation Assay, Activation Assay, Expressing, IF-cells, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Knockdown, Transduction, Control, Lysis

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 3. Neural Precursors Express ErbB4 JMa, which, When Activated, Promotes the Formation and Nuclear Translocation of an E4ICD/TAB2/N-CoR Complex (A) NRG1 treatment (5 min) induces ErbB4 phosphorylation in neural precursors (NPs). (B) NRG1 stimulation promotes E4ICD release from NPs. Cytoplasmic fractions of NPs either left untreated or stimulated with NRG1 were subjected to western blot with ErbB4 antibodies. GADPH was used to control for loading. (C) NRG1 induces ErbB4 JMa interactions with endogenous TAB2 and N-CoR in NPs. Cells were either left untreated or stimulated with NRG1 (30 min), lysed, immunoprecipitated with ErbB4 antibodies, and immunoblotted with N-CoR or TAB2 antibodies. Whole-cell lysates were blotted with the same antibodies to show the input. (D)NRG1treatment(2hr)stimulatesnucleartranslocationofendogenousE4ICD,TAB2,andN-CoRinNPsinapresenilin-dependentfashion(*p<0.01). (E) Representative images of untreated NPs (control) or NPs stimulated with NRG1 stained with ErbB4, TAB2, or N-CoR (green). Nuclei are labeled with Hoechst (blue). (F) RNAi knockdown of TAB2 inhibits NRG1-dependent N-CoR nuclear translocation, while E4ICD nuclear shuttling is not affected. NPs were trans- duced with control or TAB2 RNAi-expressing lentiviruses. Three days later, cells were treated and analyzed as in (D). Identical results were obtained with two different RNAi constructs against TAB2 (*p < 0.01).

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Translocation Assay, Phospho-proteomics, Western Blot, Control, Immunoprecipitation, Staining, Labeling, Knockdown, Expressing, Construct

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 4. NRG1-ErbB4 Signaling Inhibits Astrogenesis in a Presenilin-Dependent Fashion (A) NRG1 blocks CNTF-induced differentiation of NPs into astrocytes in a presenilin-dependent manner. CNTF treatment (2 days) induced NPs to differentiate into GFAP-expressing astrocytes. NRG1 treatment did not change the number of GFAP+ cells but significantly reduced the CNTF-mediated astrogenesis if applied 3 hr prior to CNTF. The NRG1 inhibition of CNTF-induced astrogenesis was blocked by addition of DAPT 30 min prior to the other treatments (*p < 0.05). (B) NRG1 has no effect on PDGF-induced neurogenesis. PDGF treat- ment (4 days) increased the number of cells expressing the neuronal marker b-tubulin III. Addition of NRG1 alone or before PDGF did not alter the number of b-tubulin III+ cells. (C and D) ErbB4 or TAB2 knockdown blocks NRG1 inhibition of astro- genesis. NPs were transduced with control or TAB2 or ErbB4 RNAi-ex- pressing lentiviruses. Three days later, cells were treated with CNTF and/or NRG1 and analyzed as in (A). Similar results were obtained with two different RNAi constructs against ErbB4 or TAB2 (*p < 0.05).

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Expressing, Inhibition, Marker, Knockdown, Transduction, Control, Construct

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 5. NRG1-ErbB4 Nuclear Signaling Inhibits GFAP Promoter Activation and Induces E4ICD Recruitment to this Promoter (A) NRG1 blocks CNTF-induced GFAP promoter activation in a presenilin-dependent manner. NPs were cotransfected with GFAP-luciferase and TK-renilla reporters. The next day, cells were treated with NRG1, CNTF, and/or DAPT. DAPT was added 30 min prior to NRG1. NRG1 was added 3 hr prior to CNTF. Six hours after CNTF addition, NPs were lysed and luciferase activity was measured (*p < 0.05). (B) NRG1-mediated inhibition of CNTF-induced GFAP promoter activation depends on ErbB4. NPs were transduced with control or ErbB4 RNAi-ex- pressing lentiviruses. After 3 days, cells were transfected, treated with NRG1 and/or CNTF, and analyzed as in (A). Similar results were obtained with two different RNAi constructs against ErbB4 (*p < 0.05). (C) Only the cleavable isoform HER4 JMa rescues the ErbB4 knockdown phenotype. ErbB4 endogenous to NPs was knocked down by RNAi. After 3 days, the human ErbB4 juxtamembrane isoforms (HER4 JMa or JMb) or the presenilin-resistant HER4 JMa V673I, which are resistant to the RNAi, were transfected along with the reporters. Cells were then treated and processed as in (A) (*p < 0.05). (D) Expression of activated E4ICD is sufficient to inhibit CNTF-induced GFAP promoter activation. NPs were cotransfected with empty vector, WT, or KD LexA-E4ICD plasmids along with the reporters. The next day, cells were treated with CNTF and processed as in (A). Only wild-type E4ICD blocked CNTF induction of the GFAP promoter; this blockade was presenilin independent (*p < 0.05). (E) NRG1-mediated inhibition of CNTF-induced GFAP promoter activation depends on TAB2. NPs were transduced with control or TAB2 RNAi- expressing lentiviruses. After 3 days, cells were transfected, treated, and analyzed as in (A). Similar results were obtained with two different RNAi constructs against TAB2 (*p < 0.05). (F) NRG1-mediated inhibition of CNTF-induced GFAP promoter activation requires the TAB2 C terminus. TAB2 (full-length or DC truncated) was transfected along with the reporters. Cells were then treated and processed as in (A) (*p < 0.05). (G) NRG1 induces E4ICD binding to the GFAP and S100b promoters in a presenilin-dependent manner. NPs were incubated for 30 min with vehicle or DAPT before NRG1 treatment. After 6 hr, ChIP assays were performed with antibodies to ErbB4 or control IgG. PCR primers specific for GFAP or S100b promoters were used. Primers against the first exon of HES1 serve as a negative control.

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Activation Assay, Luciferase, Activity Assay, Inhibition, Transduction, Control, Transfection, Construct, Knockdown, Expressing, Plasmid Preparation, Binding Assay, Incubation, Negative Control

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 6. Precocious Astrogenesis in ErbB4 Knockout Developing Cortex (A) Increased GFAP mRNA expression in E17.5 ErbB4/ HER4heart mouse cortex. In situ hybridization on coronal sections showing GFAP mRNA expression in ErbB4+/and ErbB4/ HER4heart littermates. No signal was generated using a sense probe. lv = lateral ventricle. Bar = 50 mm. (B) Increased GFAP protein expression in brains of E17.5 ErbB4/ HER4heart mice. GFAP immunoblot of whole-brain lysates of ErbB4/ HER4heart

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Knock-Out, Expressing, In Situ Hybridization, Generated, Western Blot

Journal: Cell

Article Title: Presenilin-dependent ErbB4 nuclear signaling regulates the timing of astrogenesis in the developing brain.

doi: 10.1016/j.cell.2006.07.037

Figure Lengend Snippet: Figure 7. Mechanism for the Regulation of NP Fate by ErbB4 Nuclear Signaling ErbB4 activation by NRG1 promotes receptor phosphorylation and its association with TAB2 and N-CoR. Ligand-induced ErbB4 cleavage by TACE and presenilin allows for the nuclear translocation of the E4ICD/ TAB2/N-CoR complex. This complex binds to specific promoters (GFAP and S100b), leading to their transcriptional repression and pre- venting their activation by factors that induce astrocyte differentiation, such as CNTF.

Article Snippet: For immunoprecipitations, lysates were precleared with agarose-conjugated normal IgG for 30 min and immunoprecipitated with agarose-conjugated ErbB4 antibody (Santa Cruz) or FLAG antibody (M2, Sigma) overnight at 4 C. Samples were washed four times with lysis buffer before the beads were resuspended in SDS sample buffer and boiled for 2 min.

Techniques: Activation Assay, Phospho-proteomics, Translocation Assay