Journal: Frontiers in Cell and Developmental Biology

Article Title: Poly (ADP-Ribose) Polymerase 1 Regulates Cajal–Retzius Cell Development and Neural Precursor Cell Adhesion

doi: 10.3389/fcell.2021.693595

Figure Lengend Snippet: Reelin induces Dab1 phosphorylation in NPCs. (A) Western blot of medium conditioned by HEK293T cells transfected with a Reelin-expressing plasmid or empty vector (pcDNA3) and their cell lysates indicates expression and secretion of Reelin. Lysates and conditioned medium (CM) were collected 24 h after transfection. Arrows indicate full-length (FL) Reelin and NR2 and NR6 fragments. (B) WT NPCs treated with 200 μL (+) or 400 μL (++) conditioned medium (CM) from Reelin-transfected HEK293T cells for 10 min have increased Dab1 phosphorylation (pDab1). (C) A total of 20 min treatment with CM from WT NPC cultures increases Dab1 phosphorylation in WT NPCs ( n = 4 separate cultures). Pretreatment with Brefeldin A (BFA; 0.75 μg/mL) for 3 h prior to CM treatment reduces Dab1 phosphorylation in WT NPCs, indicating that the state of Dab1 phosphorylation depends on the ability of cells to secrete proteins. Addition of BFA to CM after harvesting does not affect pDab1 induction. Relative band density was normalized to GAPDH. Fold change was 2.19 with p = 0.023 by one-sample t -test.

Article Snippet: The following primary antibodies and concentrations were used: mouse anti-Reelin (1:2000, Millipore #MAB5364), mouse anti-PAR (1:1000, Trevigen #4335), mouse anti-GAPDH (1:2000, ThermoFisher #MA5-15738), rabbit anti-PARP1 (1:2000, Cell Signaling Technology #9532) and rabbit anti-pDab1 (1:1000, Cell Signaling Technology #3325).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: Disabled 1 (Dab1) interacts with epidermal growth factor receptor (EGFR). ( A ) Sequence alignment of human/murine apolipoprotein E receptor 2 (ApoER2), very low density lipoprotein receptor (VLDLR) and EGFR intracellular domains was generated with Clustal Omega. NPxY motifs present in the intracellular domains of the receptors are marked by red boxes. “*” residues are identical in all sequences in the alignment, “:” conserved substitutions, “.” semi-conserved substitutions. ( B ) Western blot analysis of protein extracts from HEK293 cells expressing ApoER2 and Dab1 (Lane 1, input) or EGFR and Dab1 (Lane 4, input) which were subjected to immunoprecipitations using an antibody specific to ApoER2 (Lane 2) or EGFR (Lane 5) or an unrelated rabbit IgG antibody (Lane 3) or mouse IgG antibody (Lane 6). In upper panels Dab1 levels were detected by using a Dab1 specific antibody (Ab D4). The blots were stripped and re-probed with an ApoER2 or EGFR specific antibody (lower panels) as a pulldown control. ( C ) Murine embryonic neurons (E15.5) were lysed and the protein extract was subjected to immunoprecipitation using an antibody specific to ApoER2 (Lane 2) or EGFR (Lane 5) or unrelated rabbit (Lane 3) or mouse IgG (Lane 6) as a control. The extracts and precipitates were analyzed by western blotting. The vertical line indicates that the blot was spliced. ( D ) HEK293 cells expressing fluorescently tagged variants of ApoER2 ( j ), EGFR ( a , d ) and Dab1 ( b , k , n ) or fluorophores alone; mGFP ( e , h ) or mCherry ( g , m ) were imaged by confocal fluorescence microscopy. Merged images are presented in ( c , f , i , l , o ). Scale bar represents 20 µm.

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Sequencing, Generated, Western Blot, Expressing, Immunoprecipitation, Fluorescence, Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: EGF induces Dab1 phosphorylation. Western blot analysis of protein extracts from HEK293 ( A ) and NIH3T3 ( C ) cells expressing EGFR and Dab1. Cells were either left untreated (Lane 1) or treated with human EGF (10 ng/mL) for 3 (Lane 2), 10 (Lane 3), or 20 min (Lane 4). Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Dab1 phosphorylation levels (Panel A1/C1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blots were stripped and re-probed for Dab1 using Ab D4 (panel A2/C2). Extracts were blotted with an antibody specific for phosphorylated EGFR (Tyr 1173) (panel A3/C3). As a loading control, GAPDH was used (Panel A4/C4). Anti-phosphotyrosine signal was normalized to the anti-Dab1 signal and relative levels of phosphorylated Dab1 from 4 independent experiments in HEK293 ( B ) and NIH3T3 ( D ) are presented. The levels of Dab1 phosphorylation in the untreated controls were set to 1. Data were analyzed by an unpaired, two-tailed t -test. ( E ) Primary neuronal cultures (DIV7) were treated with Reelin conditioned medium (RCM, Lane 2), Mock conditioned medium (MCM, Lane 1) for 20 min or with 20 ng/mL murine EGF for 3 min (Lane 4) or left untreated (Lane 3). Cells were lysed and the protein extracts were subjected to immunoprecipitation using an antibody specific to Dab1 (Ab 54). Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blot was stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for phosphorylated EGFR (Tyr 1173) (Panel 3), total levels of EGFR (Panel 4). As a neuronal loading control, Ab NeuN was used (Panel 5). The extracts and precipitates were analyzed by western blotting. Bands were scanned with ChemiDoc Touch Imaging System (BioRad), the anti-phosphotyrosine signal was normalized to the anti-Dab1 band and relative levels of phosphorylated Dab1 from 3–4 independent experiments are presented. The levels of Dab1 phosphorylation in the untreated or mock controls were set to 1. Relative intensity of the presented blot is shown in a box above the first panel. Data were analyzed by way of an unpaired, two-tailed t -test, * p ≤ 0.05, ** p ≤ 0.01, ns, not significant; dotes, number of experiments. Error bars represent standard deviation.

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Western Blot, Expressing, Immunoprecipitation, Two Tailed Test, Imaging, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: Dab1 phosphorylation depends on EGFR kinase activity. Western blot analysis of protein extracts from HEK293 ( A ) and NIH3T3 ( B ) cells expressing EGFR and Dab1. Inhibitors of EGFR phosphorylation, Gefitinib (Lane 3) and Neratinib (Lane 4) were added 2 h before EGF treatment (3 min at 37 °C). Lane 1 represents untreated negative controls, Lane 2 represents the EGF treated positive controls. Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). ( C ) Western blot analysis of protein extracts HEK293T expressing Dab1 and either a wild type (WT)-like EGFR or a kinase-dead (KD) EGFR. Lanes 1 and 3 represent the untreated negative controls and Lanes 2 and 4 represent EGF treatment. Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Bands were scanned with ChemiDoc Touch Imaging System (BioRad, Hercules, CA, USA) and the anti-phosphotyrosine signal was normalized to the anti-Dab1 band. The level of Dab1 phosphorylation in the untreated WT EGFR (lane 1) control was set to 1. Relative intensity is shown in a box above the first panel. Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blots were stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for phosphorylated EGFR (Tyr 1173) (Panel 3) and EGFR (C, Panel 4). As a loading control GAPDH was used (Panel 4 in A and B or 5 in C).

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Activity Assay, Western Blot, Expressing, Immunoprecipitation, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: Presence of ApoER2 diminishes EGF-mediated Dab1 phosphorylation. Western blot analysis of HEK293 ( A ) cells expressing EGFR and Dab1 (Lanes 1 and 2, ED) or ApoER2, EGFR and Dab1 (Lanes 3 and 4, AED). Cells were either left untreated (Lanes 1 and 3) or treated with human EGF (10 ng/mL) for 3 min (Lanes 2 and 4). Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blots were stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for EGFR phosphorylation (Tyr 1173) (Panel 3) and GAPDH was used as a loading control (Panel 4). ( B ) The anti-phosphotyrosine signal was normalized to the anti-Dab1 signal and relative levels of phosphorylated Dab1 from 4 independent experiments in HEK293 are presented. The level of Dab1 phosphorylation in the untreated control was set to 1. Data were analyzed by an unpaired, two-tailed t -test, *** p ≤ 0.001; dots, number of experiments. Error bars represent standard deviation.

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Western Blot, Expressing, Immunoprecipitation, Two Tailed Test, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: Cell proliferation rate is decreased in Dab1 k.o. cells. ( A ) Validation of HEK293 Dab1 k.o. cells. RNA from HEK293 wt and HEK293 Dab1 k.o. cells was extracted and reverse transcribed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) using oligo (dT) 18 primers. Subsequently, PCR using primers specific for complete cDNA sequence of human Dab1 was performed and the presence of the band with the expected size of about 1700 bp corresponding to human Dab1 (Lane 1) was analyzed by agarose gel electrophoresis. As positive control a plasmid carrying the Dab1 sequence was used (Lane 5). Negative controls used in this study: HEK293 w/o RT, no reverse transcriptase (Lane 2); NTC, no RNA template (Lane 3); PCR neg, no cDNA template (Lane 6). ( B ) HEK293 wt, Dab1 k.o. cells, and Dab1 k.o. cells expressing Dab1_555 were treated with EdU (10 µM) and incubated for 2 h at 37 °C. Cells were fixed and EdU incorporated DNA was stained via Click-iT assay with AlexaFluor488 and subsequently with DAPI. Pictures of 10 different fields of view were taken and analyzed by Image J. Scale bar represents 100 µm. EdU and DAPI stained nuclei were counted and set into relation. Data were analyzed by one-way ANOVA multiple comparison test. ( C ) Proliferation/survival WST-1 assay performed in HEK293 wt and Dab1 k.o. cells transfected with pClneo or Dab1_555. WST-1 (10 nM) was added to each well and absorbance (480/30 nm) was measured after 2 h. The data were derived from two independent experiments, which were performed with 7 replicates. Data were analyzed by one-way ANOVA multiple comparison test, ** p ≤ 0.01, **** p ≤ 0.0001, ns, not significant, dots, number of experiments.

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Sequencing, Agarose Gel Electrophoresis, Positive Control, Plasmid Preparation, Expressing, Incubation, Staining, WST-1 Assay, Transfection, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: EGF induces phosphorylation of the Dab1 Early isoform. ( A ) Embryonic primary neurons (DIV3) and intestinal epithelial cells (IECs) were lysed and the protein extracts were analyzed by western blotting for expression of Dab1 (Lanes 1 and 2), EGFR (Lanes 3 and 4), VLDLR (Lanes 5 and 6), and ApoER2 (Lanes 7 and 8). Phosphorylation level of Dab1 in IECs was analyzed by Dab1 immunoprecipitation and probing eluates samples with phosphotyrosine specific antibody (Lanes 9 and 10, Panel 1). Blot was stripped and re-probed with a Dab1 specific antibody (Lanes 9 and 10, Panel 2). As negative control unrelated mouse IgG was used. ( B ) Western blot analysis of HEK293 cells (Lanes 1 and 2) and HEK293 cells overexpressing EGFR and chDab1_E (Lanes 3 and 4). Cells were either left untreated (Lanes 1 and 3) or treated with human EGF (10 ng/mL) for 3 min (Lanes 2 and 4). Immunoprecipitation using a Dab1 specific antibody (Ab 54) was performed. Dab1 phosphorylation levels (Panel 1, Lanes 1–4) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blot was stripped and re-probed for Dab1 using Ab D4 (Panel 2). Extracts were blotted with an antibody specific for EGFR phosphorylation (Panel 3, Lanes 1–4) and total levels of EGFR (Panel 4, Lanes 1–4). Extracts were also blotted with an antibody specific for Dab1 phosphorylation (Tyr 232) (Panel 1, Lanes 5–8). The blot was stripped and then re-probed with a Dab1 specific antibody (Panel 2, Lanes 5–8). ( C ) Western blot analysis of HEK293 cells expressing ApoER2 and Dab1_555 (Lanes 1 and 2) or chDab1_E (Lanes 3 and 4). Cells were either treated with Reelin (Lanes 2 and 4) or Mock conditioned medium (Lanes 1 and 3) for 20 min. Immunoprecipitation using a Dab1 specific antibody was performed (Ab 54). Dab1 phosphorylation levels (Panel 1) were detected using an antibody against phosphorylated tyrosine residues (Ab PY99). The blot was stripped and re-probed for Dab1 using Ab D4 (panel 2).

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Western Blot, Expressing, Immunoprecipitation, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Disabled 1 Is Part of a Signaling Pathway Activated by Epidermal Growth Factor Receptor

doi: 10.3390/ijms22041745

Figure Lengend Snippet: The following antibodies were used in this study at the indicated dilutions.

Article Snippet: Phospho-Dab1 (Tyr232) Antibody , #3325 , Cell Signaling Technology (Danvers, MA, USA) , WB 1:500.

Techniques: Produced

Journal: Frontiers in Molecular Neuroscience

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy

doi: 10.3389/fnmol.2019.00053

Figure Lengend Snippet: The following antibodies were used in this study at the indicated dilutions.

Article Snippet: Phospho-Dab1 (Tyr232) , #3325 , Cell Signaling , WB 1:1,000.

Techniques:

Journal: Frontiers in Molecular Neuroscience

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy

doi: 10.3389/fnmol.2019.00053

Figure Lengend Snippet: Central fragment of Reelin (R3–6) does not induce Dab1 phosphorylation. HEK293 cells expressing Dab1_myc and ApoER2 (A) or Dab1_myc and VLDLR (B) or Dab1_myc, ApoER2, and VLDLR (C) were kept in Imaging Medium for 30 min. Next, the medium was changed to RCM containing full length Reelin (FL Rln, lanes 2) or R3–6 (lanes 3) was added to the cells (final concentration, 30 nM) or cells were left untreated (neg.ctrl, lanes 1). After 20 min incubation at 37°C, cells were washed and lysed in NP-40 lysis buffer. Lysates were analyzed by western blotting using phospho-Dab1 antibody (Ab pDab1) and GAPDH antibody. Membranes were stripped and subsequently re-probed for the detection of Dab1 (AbD4). ApoER2 was detected by Ab20 and VLDLR by AbAF2258.

Article Snippet: Phospho-Dab1 (Tyr232) , #3325 , Cell Signaling , WB 1:1,000.

Techniques: Expressing, Imaging, Concentration Assay, Incubation, Lysis, Western Blot

Journal: Frontiers in Molecular Neuroscience

Article Title: Differential Action of Reelin on Oligomerization of ApoER2 and VLDL Receptor in HEK293 Cells Assessed by Time-Resolved Anisotropy and Fluorescence Lifetime Imaging Microscopy

doi: 10.3389/fnmol.2019.00053

Figure Lengend Snippet: Model of ApoER2 and VLDLR mediated signaling during cortical development. Radial glia cells divide asymmetrically to produce postmitotic neurons. During early stages of cortical development these neurons migrate via somal translocation. When the thickness of the cerebral wall increases neurons switch their migration mode to multipolar migration followed by locomotion. Thus, polarized neurons leave the ventricular (VZ) and subventricular zone (SVZ), depolarize and migrate through the IZ via a multipolar migration mode. The depolarization step is ill defined and may involve ApoER2 (A)-mediated signaling driven by the central Reelin fragment R3–6 (box 1). Repolarization of the neurons as prerequisite to glia cell guided locomotion is induced by Dab1 phosphorylation mediated by a netrin 1—deleted in colon cancer (DCC) signaling cascade and/or by the canonical Reelin pathway (box 2). Locomotion and terminal translocation is orchestrated by a complex set of Reelin (full length, FL) signals transmitted by clustering of ApoER2 (A) and VLDLR (V) homo- oligomers, leading to Dab1 phosphorylation (box 3). CP, cortical plate; IZ, intermediate zone; MZ, marginal zone.

Article Snippet: Phospho-Dab1 (Tyr232) , #3325 , Cell Signaling , WB 1:1,000.

Techniques: Translocation Assay, Migration