Journal: bioRxiv

Article Title: Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

doi: 10.1101/2022.01.13.476283

Figure Lengend Snippet: (A) HeLa cells transfected with TRP120-GFP (green) and probed for endogenous Notch-1 (red) demonstrate colocalization by immunofluorescent microscopy. Colocalization was quantitated by Pearson’s and Mander’s coefficient (−1 no colocalization; +1 strong colocalization). (B and C) His-tag pull down assays demonstrating direct interaction between TRP120 and Notch-1. Recombinant Fc-tagged Notch-1 LBR was incubated with (B) TRP120-FL-His, (C) TRP120-TR-His or TRX-His negative control on Talon metal affinity resin. Bound Notch-1, TRP120-His, α-TRP120 against a TR peptide or TRX-His were detected with α-Notch-1, α-TRP120 or α-TRX antibodies. (D-F) Surface plasmon resonance of (D) TRP120-FL-His, (E) TRP120-TR-His or (F) TRX-His with Fc-tagged Notch-1 LBR on a Biacore T100 with a series S Ni-nitrilotriacetic acid (NTA) sensor chip. TRP120-FL-His, TRP120-TR-His or TRX-His were immobilized on the NTA chip and 2-fold dilutions (800nM to 25nM) of Fc-tagged Notch-1 LBR were used as analyte to determine binding affinity (K D ). Sensograms and K D are representative of data from triplicate experiments. (G) THP-1 cells were treated with rTRX- or rTRP120-FL-coated fluorescent microspheres for varying time points (5-60 mins). Colocalization was visualized by confocal immunofluorescent microscopy. Notch-1 was immunostained with tetramethylrhodamine isothiocyanate [TRITC] and TRP120-coated fluorescein isothiocyanate [FITC] auto-fluorescent microspheres. Nuclei were stained with DAPI (blue). White boxes indicate areas of colocalization measurements. Scale bar = 10 μm. (H) Dot blot of PBS, TRX or TRP120-FL-coated microspheres probed with α-TRX or α-TRP120 antibodies, respectively.

Article Snippet: Recombinant His-tagged TRP120 (10 μg) and Notch-1 (10 μg) (Sino Biological) were incubated with Ni-NTA beads alone, or in combination, for 4 h at 4°C.

Techniques: Transfection, Microscopy, Recombinant, Incubation, Negative Control, SPR Assay, Binding Assay, Staining, Dot Blot

Journal: bioRxiv

Article Title: Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

doi: 10.1101/2022.01.13.476283

Figure Lengend Snippet: (A) Overlapping TRP120-TR IDD peptide sequences (P1-P3) (B) THP-1 cells or (C) Primary human monocytes were incubated with synthetic TRP120-TR IDD peptides to determine the TRP120-TR Notch-1 memetic motif responsible for Notch activation. TRP120-TR peptides were overlapping peptides spanning an entire TR domain. Cells were treated with peptide (1 μg/ml) for 2 h and confocal immunofluorescent microscopy was used to visualize NICD localization. NICD nuclear translocation denotes Notch activation. A scrambled peptide (Ctrl-p) was used as negative control and E.ch . infected cells were used as positive control. To determine if direct interaction of the TRP120-N1-P3 peptide and Notch receptor was necessary for Notch activation, THP-1 cells were pre-treated with DAPT, a γ-secretase inhibitor, and treated with TRP120-N1-P3 peptide for 2 h.

Article Snippet: Recombinant His-tagged TRP120 (10 μg) and Notch-1 (10 μg) (Sino Biological) were incubated with Ni-NTA beads alone, or in combination, for 4 h at 4°C.

Techniques: Incubation, Activation Assay, Microscopy, Translocation Assay, Negative Control, Infection, Positive Control

Journal: bioRxiv

Article Title: Ehrlichia SLiM ligand mimetic activates Notch signaling in human monocytes

doi: 10.1101/2022.01.13.476283

Figure Lengend Snippet: (A) TRP120-N1 SLiM (P4-P6) and mutant (dmut) peptide sequences. (B) THP-1 cells or (C) primary human monocytes were treated with synthetic TRP120-TR SLiM peptides to identify the TRP120-TR Notch-1 SLiM memetic motif. TRP120-TR peptides were SLiM peptides spanning the entire TRP120-N1-P3 peptide sequence. TRP120-N1-P3 mutant peptide (dmut) has a deletion of the TRP120-N1-P6 amino acids. Cells were treated with peptide (1 μg/ml) for 2 h and NICD localization visualized by confocal microscopy. TRP120-N1-P3 peptide was used as a positive control. To determine if direct interaction of the TRP120-N1-P6 peptide and Notch receptor was necessary for Notch activation, THP-1 cells were pre-treated with DAPT, a γ-secretase inhibitor, and treated with TRP120-N1-P6 peptide for 2 h. Representative data of all experiments are shown (n = 3).

Article Snippet: Recombinant His-tagged TRP120 (10 μg) and Notch-1 (10 μg) (Sino Biological) were incubated with Ni-NTA beads alone, or in combination, for 4 h at 4°C.

Techniques: Mutagenesis, Sequencing, Confocal Microscopy, Positive Control, Activation Assay

Journal: Nucleic Acids Research

Article Title: GSK3β is a negative regulator of the transcriptional coactivator MAML1

doi: 10.1093/nar/gkp724

Figure Lengend Snippet: GSK3β directly interacts with the MAML1 N-terminus. ( A ) Schematic diagram of the MAML1 protein. GST-tagged MAML1 proteins as indicated, were incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β ( B ) or recombinant affinity-purified GSK3β ( C ), and MAML1-interacting GSK3β detected with immunoblot. ( D ) Recombinant HIS-GSK3β and GST-MAML1 proteins were affinity-purified and incubated with [γ- 32 P]ATP. Proteins were separated by SDS–PAGE and visualized by autoradiography. ( E ) GST-MAML1 proteins were incubated with HIS-GSK3β in the presence of cold ATP. The kinase reactions were separated by SDS–PAGE and stained with Pro-Q Diamond phosphoprotein staining kit. ( F ) Vectors expressing GSK3β and were cotransfected with a luciferase reporter containing five GAL4 binding sites into U2OS cells.

Article Snippet: In GST-MAML1 interaction assays, ∼2 µg of purified GST-tagged MAML1 proteins, bound to 20 µl of glutathione-Sepharose beads, was incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and diluted 3 times with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 10% glycerol, 0.1% NP-40, 1 mM DTT and 1 mM PMSF) ( B) or incubated with HIS-GSK3β (Invitrogen) and FLAG-Notch1 ICD (as indicated in E, C and D), expressed in Sf9 ( Spodoptera frugiperda ) cells via baculovirus and purified as described in ref. ( ).

Techniques: Incubation, Transfection, Recombinant, Affinity Purification, Western Blot, SDS Page, Autoradiography, Staining, Expressing, Luciferase, Binding Assay

Journal: Nucleic Acids Research

Article Title: GSK3β is a negative regulator of the transcriptional coactivator MAML1

doi: 10.1093/nar/gkp724

Figure Lengend Snippet: GSK3β decreases MAML1 activity. ( A ) HEK-293 cells were cotransfected with a luciferase reporter containing five GAL4 binding sites and vectors expressing GAL4-MAML1 and GSK3β as indicated. ( B ) GAL4-MAML1 was cotransfected into HEK-293 cells with the luciferase reporter in the absence or presence of SB41. ( C ) HEK-293 cells were transfected with a vector expressing FLAG-MAML1, MAML1 was affinity-purified with M2-agarose, and proteins separated by SDS–PAGE. Immunoblot detected MAML1, and GSK3β protein that interacted with MAML1. ( D ) Endogenous MAML1 protein was immunoprecipitated from C33A cells with an antibody recognizing the N-terminus of MAML1. Proteins were separated by SDS–PAGE and detection of MAML1, and MAML1-interacting proteins, was monitored by immunoblot with antibodies recognizing MAML1 and GSK3β. ( E ) GST-MAML1 and GST coupled to glutathione-Sepharose beads were incubated with affinity-purified HIS-GSK3β, and MAML1-interacting GSK3β was monitored by immunoblot. The input represents 10% of GSK3β used in the binding reaction. ( F ) GST-GSK3β and GST coupled to glutathione-Sepharose beads were incubated with affinity-purified FLAG-MAML1, and MAML1 binding to GSK3β was monitored by immunoblot. The input represents 10% of the MAML1 used in the binding reaction. ( G ) GSK3β phosphorylates MAML1 in vitro . Recombinant affinity-purified proteins HIS-GSK3β, GST-MAML1 or GST, were incubated with [γ- 32 P]ATP, in the presence or absence of SB41, proteins were separated by SDS–PAGE and visualized by autoradiography.

Article Snippet: In GST-MAML1 interaction assays, ∼2 µg of purified GST-tagged MAML1 proteins, bound to 20 µl of glutathione-Sepharose beads, was incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and diluted 3 times with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 10% glycerol, 0.1% NP-40, 1 mM DTT and 1 mM PMSF) ( B) or incubated with HIS-GSK3β (Invitrogen) and FLAG-Notch1 ICD (as indicated in E, C and D), expressed in Sf9 ( Spodoptera frugiperda ) cells via baculovirus and purified as described in ref. ( ).

Techniques: Activity Assay, Luciferase, Binding Assay, Expressing, Transfection, Plasmid Preparation, Affinity Purification, SDS Page, Western Blot, Immunoprecipitation, Incubation, In Vitro, Recombinant, Autoradiography

Journal: Nucleic Acids Research

Article Title: GSK3β is a negative regulator of the transcriptional coactivator MAML1

doi: 10.1093/nar/gkp724

Figure Lengend Snippet: The MAML1-Notch1 ICD interaction is not abolished by GSK3β. ( A ) Vectors expressing FLAG-Notch1 ICD, HA-GSK3β-S9A and MAML1 1–1016 were cotransfected into Cos7 cells, and after 24 h cells were immunostained with antibodies recognizing the FLAG- or HA-tags or MAML1. ( B ) HEK-293 cells were cotransfected with a luciferase reporter containing five GAL4 binding sites and vectors expressing GAL4-Notch1 ICD, MAML1 and GSK3β. ( C ) C33A cells were transfected with Notch1 ICD and siRNA MAML1 or control (ctrl) siRNA, and incubated in the presence or absence of SB41. Expression of MAML1, GAPDH and the Notch target gene Hes1 was analyzed with immunoblot with antibodies recognizing MAML1, Hes1 and GAPDH. ( D ) GST-MAML1 and GST were incubated, in the presence or absence of ATP, with affinity-purified HIS-GSK3β and FLAG-Notch1 ICD proteins as indicated, and MAML1-interacting proteins were detected with immunoblot.

Article Snippet: In GST-MAML1 interaction assays, ∼2 µg of purified GST-tagged MAML1 proteins, bound to 20 µl of glutathione-Sepharose beads, was incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and diluted 3 times with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 10% glycerol, 0.1% NP-40, 1 mM DTT and 1 mM PMSF) ( B) or incubated with HIS-GSK3β (Invitrogen) and FLAG-Notch1 ICD (as indicated in E, C and D), expressed in Sf9 ( Spodoptera frugiperda ) cells via baculovirus and purified as described in ref. ( ).

Techniques: Expressing, Luciferase, Binding Assay, Transfection, Incubation, Western Blot, Affinity Purification

Journal: Nucleic Acids Research

Article Title: GSK3β is a negative regulator of the transcriptional coactivator MAML1

doi: 10.1093/nar/gkp724

Figure Lengend Snippet: Active GSK3β regulates MAML1 transcription. ( A ) Vectors expressing GAL4-MAML1 and HA-GSK3β as WT, S9A or K85R, were cotransfected into HEK-293 cells with a luciferase reporter. ( B ) Vectors expressing MAML1 and GSK3β were cotransfected into HEK-293 cells as indicated, whole-cell extracts were prepared, and MAML1 protein immunoprecipitated. Proteins were separated by SDS–PAGE and detection of MAML1, and MAML1-interacting proteins, was monitored by immunoblot with antibodies recognizing MAML1 and GSK3β.

Article Snippet: In GST-MAML1 interaction assays, ∼2 µg of purified GST-tagged MAML1 proteins, bound to 20 µl of glutathione-Sepharose beads, was incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and diluted 3 times with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 10% glycerol, 0.1% NP-40, 1 mM DTT and 1 mM PMSF) ( B) or incubated with HIS-GSK3β (Invitrogen) and FLAG-Notch1 ICD (as indicated in E, C and D), expressed in Sf9 ( Spodoptera frugiperda ) cells via baculovirus and purified as described in ref. ( ).

Techniques: Expressing, Luciferase, Immunoprecipitation, SDS Page, Western Blot

Journal: Nucleic Acids Research

Article Title: GSK3β is a negative regulator of the transcriptional coactivator MAML1

doi: 10.1093/nar/gkp724

Figure Lengend Snippet: MAML1 directs GSK3β to nuclear bodies. ( A ) Vectors expressing FLAG-MAML1 domains as indicated, and HA-GSK3β were cotransfected into Cos7 cells, and after 24 h the cells were immunostained with antibodies recognizing the FLAG- and HA-tags. ( B ) Vectors expressing HA-GSK3β-S9A, HA-GSK3β-K85R and FLAG-MAML1 were cotransfected into Cos7 cells, and cells were immunostained after 24 h with antibodies recognizing the FLAG- and HA-tags. ( C ) Cos7 cells were transfected with FLAG-MAML1 (or empty vector) and immunostained after 24 h with antibodies recognizing the FLAG-tag, and endogenous GSK3β phosphorylated at serine 9 (P-S9).

Article Snippet: In GST-MAML1 interaction assays, ∼2 µg of purified GST-tagged MAML1 proteins, bound to 20 µl of glutathione-Sepharose beads, was incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and diluted 3 times with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 10% glycerol, 0.1% NP-40, 1 mM DTT and 1 mM PMSF) ( B) or incubated with HIS-GSK3β (Invitrogen) and FLAG-Notch1 ICD (as indicated in E, C and D), expressed in Sf9 ( Spodoptera frugiperda ) cells via baculovirus and purified as described in ref. ( ).

Techniques: Expressing, Transfection, Plasmid Preparation, FLAG-tag

Journal: Nucleic Acids Research

Article Title: GSK3β is a negative regulator of the transcriptional coactivator MAML1

doi: 10.1093/nar/gkp724

Figure Lengend Snippet: GSK3β inhibits MAML1-dependent global histone acetylation. f-MAML1 or HEK-293 cells (ctrl cell line), were incubated in the presence or absence of SB41. The levels of expressed MAML1, acetylated histone H3 (Ac-H3), and tubulin, were monitored with immunoblot.

Article Snippet: In GST-MAML1 interaction assays, ∼2 µg of purified GST-tagged MAML1 proteins, bound to 20 µl of glutathione-Sepharose beads, was incubated with whole-cell extract from HEK-293 cells transfected with pCDNA-GSK3β lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and diluted 3 times with buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl, 10% glycerol, 0.1% NP-40, 1 mM DTT and 1 mM PMSF) ( B) or incubated with HIS-GSK3β (Invitrogen) and FLAG-Notch1 ICD (as indicated in E, C and D), expressed in Sf9 ( Spodoptera frugiperda ) cells via baculovirus and purified as described in ref. ( ).

Techniques: Incubation, Western Blot