rabbit anti gef h1  (Danaher Inc)

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Hierarchal clustering of MDP induced genes in BMDMs from wild-type, Pglyrp1 -/- , Nod2 -/- , or Arhgef2 -/- mice. b , c BMDMs from wild type, Pglyrp1 -/ , Nod2 -/- , or Arhgef2 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP and analyzed via western blotting with the indicated antibodies. d BMDMs from wild type or Ifnar1 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP for 18 h and analyzed for STAT1 pathway activation with phospho-specific antibodies as indicated. e Wild type or GEF-H1-deficient BMDMs were treated with 100 ng/mL of IFNγ or IFNβ for 18 h and analyzed via western blotting with the indicated antibodies. f BMDMs from wild type, Pglyrp1 -/ , and Nod2 -/- mice were stimulated with 25 μM GMTriP-K or 25 μM MDP, lysed, and analyzed via western blotting with the indicated antibodies. g qPCR analysis of gene expression in BMDMs from indicated mouse strains stimulated with 25 µM GMTriP-K or 25 µM GMDiP for 18 h. Data are presented as mean ± SEM with indicated P values analyzed by one-way ANOVA. All experiments were repeated twice and yielded consistent results.

Article Snippet: The following antibodies were used in this study: rabbit antibodies against GEF-H1 (ab155785; Abcam,1/1000 dilution), sheep anti-mouse GEF-H1 (Exalpha Biological; #X1089S, 1/1000 dilution), Rabbit anti-SEC61β (D5Q1W) (Cell Signaling,#14648,1:1000 dilution), Mouse anti-GM130 (BD biosciences #610822, 1/1000 dilution), GAPDH (Cell signaling, #2118, 1:500 dilution), PGLYRP1(R&D system, #AF2696, 1:500 dilution), NOD2 (Invitrogen, #PA5-104317, 1:500 dilution), RB anti-phospho-stat1(Tyr701) (58D6) (Cell signaling, #9197,1/1000 dilution), RB anti-stat1 (D1K9Y) (Cell signaling, # 9172, 1/1000 dilution), and mouse anti-β-actin (8H10D10) (Cell signaling, #3700, 1/10000 dilution), Anti-FLAG (Millipore Sigma, #F7425, 1/3000 dilution).

Techniques: Western Blot, Activation Assay, Expressing

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: a Western blot analysis of protein expression in BMDMs from wildtype, Arhgef2-/- , Pglyrp1-/- , and Nod2-/- mice. b Time course of protein interactions with GEF-H1 after GMTriP-K stimulation of BMDMs. c Analysis of protein interactions with PGLYRP1 or d NOD2 after GMTriP-K stimulation of BMDMs. e Input assessment of protein expression for the IPs in ( c , d ). f Assessment of protein interactions with GEF-H1 after stimulation with indicated immune stimuli. Representative experiments of at least three repeats are shown.

Article Snippet: The following antibodies were used in this study: rabbit antibodies against GEF-H1 (ab155785; Abcam,1/1000 dilution), sheep anti-mouse GEF-H1 (Exalpha Biological; #X1089S, 1/1000 dilution), Rabbit anti-SEC61β (D5Q1W) (Cell Signaling,#14648,1:1000 dilution), Mouse anti-GM130 (BD biosciences #610822, 1/1000 dilution), GAPDH (Cell signaling, #2118, 1:500 dilution), PGLYRP1(R&D system, #AF2696, 1:500 dilution), NOD2 (Invitrogen, #PA5-104317, 1:500 dilution), RB anti-phospho-stat1(Tyr701) (58D6) (Cell signaling, #9197,1/1000 dilution), RB anti-stat1 (D1K9Y) (Cell signaling, # 9172, 1/1000 dilution), and mouse anti-β-actin (8H10D10) (Cell signaling, #3700, 1/10000 dilution), Anti-FLAG (Millipore Sigma, #F7425, 1/3000 dilution).

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: PGLYRP1-mediated intracellular peptidoglycan detection promotes intestinal mucosal protection

doi: 10.1038/s41467-025-57126-9

Figure Lengend Snippet: PGLYRP1 is required for detection of GMTriP-K. PGLYRP1 localizes to the endoplasmic reticulum and Golgi and interacts with NOD2 and GEF-H1 to induce the expression of immune regulators that regulate intestinal inflammation (Created in BioRender. Reinecker, H. (2025) https://BioRender.com/i55t156 ).

Article Snippet: The following antibodies were used in this study: rabbit antibodies against GEF-H1 (ab155785; Abcam,1/1000 dilution), sheep anti-mouse GEF-H1 (Exalpha Biological; #X1089S, 1/1000 dilution), Rabbit anti-SEC61β (D5Q1W) (Cell Signaling,#14648,1:1000 dilution), Mouse anti-GM130 (BD biosciences #610822, 1/1000 dilution), GAPDH (Cell signaling, #2118, 1:500 dilution), PGLYRP1(R&D system, #AF2696, 1:500 dilution), NOD2 (Invitrogen, #PA5-104317, 1:500 dilution), RB anti-phospho-stat1(Tyr701) (58D6) (Cell signaling, #9197,1/1000 dilution), RB anti-stat1 (D1K9Y) (Cell signaling, # 9172, 1/1000 dilution), and mouse anti-β-actin (8H10D10) (Cell signaling, #3700, 1/10000 dilution), Anti-FLAG (Millipore Sigma, #F7425, 1/3000 dilution).

Techniques: Expressing

Journal: Life Science Alliance

Article Title: Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation

doi: 10.26508/lsa.202402585

Figure Lengend Snippet: (A) Purified GST-RhoA(G17A) fusion protein was incubated with cell lysates without or with TBC1D3C expression. Protein complexes recovered from the beads were separated by SDS–PAGE and stained by Coomassie blue. The protein bands were analyzed by mass spectrometry. (B, C, D, E) Data-dependent acquisition analysis of the featured peptides YPLLISR and QDLTTALGLVK from GEF-H1, including their fragment ion patterns, precursor ions, and fragment ions. The peptide quantification was performed for the “control” and “TBC1D3C expressed” groups using precursor ions (middle) and fragment ions (right), respectively.

Article Snippet: Rabbit antibodies against β-actin, GAPDH, and Myc were purchased from HuaBio-Antibodies; rabbit antibodies against RhoA and GEF-H1 were purchased from Cell Signaling Technology; mouse antibodies against TBC1D3C were purchased from Santa Cruz Technology; antibodies against Tctex, DIC, and 14-3-3 were purchased from Proteintech; mouse antibody against α-tubulin was purchased from Life Technologies; goat anti-rabbit and goat anti-mouse IgG-conjugated horseradish peroxidase antibodies were purchased from Promega; and Alexa Fluor–conjugated goat anti-mouse and rabbit IgG antibodies were bought from Jackson ImmunoResearch.

Techniques: Purification, Incubation, Expressing, SDS Page, Staining, Mass Spectrometry, Control

Journal: Life Science Alliance

Article Title: Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation

doi: 10.26508/lsa.202402585

Figure Lengend Snippet: (A, B, C) TBC1D3C promotes the activation of GEF-H1. (B, C) Relative active GEF-H1 in (A). Active GEF-H1 was pulled down using GST-RhoA (G17A) fusion protein from lysates expressing TBC1D3C or TBC1D3C-RD2A. (D, E, F) Immunofluorescence staining reveals changes in actin dynamics and GEF-H1 subcellular localization in response to TBC1D3C induction or Noc (1 μM, 1 h) treatment; scale bar, 20 μm. (E, F) Quantification of F-actin in (D). TBC1D3C and TBC1D3C -RD2A U2OS TetOn cells with or without Dox or Noc treatment were transfected with GFP-GEF-H1 and subjected to immunofluorescence staining using Phalloidin-iFluor 555 Reagent. (G) Activation of RhoA induced by TBC1D3C is abolished in GEF-H1 knockout (KO) HEK293A cells. HEK293A TetOn TBC1D3C cells with or without GEF-H1 depletion were pulled down using GST-RBD fusion protein, and immunoprecipitated and whole-cell lysates were resolved by Western blotting. (H) TBC1D3C induces actin filament assembly, which is abolished by GEF-H1 depletion. HEK293A TetOn TBC1D3C cells with or without GEF-H1 depletion were induced with or without Dox and subjected to immunofluorescence staining using Phalloidin-iFluor 555 Reagent; scale bar, 20 μm. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Cntrl group; # P < 0.05, ## P < 0.01, ### P < 0.001 versus Dox, 24 h; scale bar, 10 μm.

Article Snippet: Rabbit antibodies against β-actin, GAPDH, and Myc were purchased from HuaBio-Antibodies; rabbit antibodies against RhoA and GEF-H1 were purchased from Cell Signaling Technology; mouse antibodies against TBC1D3C were purchased from Santa Cruz Technology; antibodies against Tctex, DIC, and 14-3-3 were purchased from Proteintech; mouse antibody against α-tubulin was purchased from Life Technologies; goat anti-rabbit and goat anti-mouse IgG-conjugated horseradish peroxidase antibodies were purchased from Promega; and Alexa Fluor–conjugated goat anti-mouse and rabbit IgG antibodies were bought from Jackson ImmunoResearch.

Techniques: Activation Assay, Expressing, Immunofluorescence, Staining, Transfection, Knock-Out, Immunoprecipitation, Western Blot

Journal: Life Science Alliance

Article Title: Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation

doi: 10.26508/lsa.202402585

Figure Lengend Snippet: (A) Microtubule dynamics are not affected by induction of TBC1D3C or TBC1D3C-RD2A over time. Cells were treated with Dox or together with Noc (1 μM, 1 h) or PTX (200 nM, 24 h) as indicated and stained with α-tubulin antibody; scale bar, 20 μm. (B) TBC1D3C promotes actin stress fiber formation without altering microtubule structure. Cells were treated as indicated and stained with α-tubulin antibody for microtubules and Phalloidin-iFluor 555 Reagent for actin; scale bar, 20 μm. (C, D, E) TBC1D3C induces GEF-H1 release from the microtubule without changes in microtubule dynamics; scale bar, 20 μm. (D, E) Correlation coefficients of GEF-H1 and microtubules in (C). Cells were treated as indicated with GFP-GEF-H1 transfection for GEF-H1 labeling, and stained with α-tubulin antibody for microtubules. (F) TBC1D3C inhibits the binding of GEF-H1 to Tctex, DIC, and 14-3-3 protein complexes. Cells treated as indicated were subjected to immunoprecipitation with GEF-H1 antibody, and immunoprecipitated and whole-cell lysate proteins were immunoblotted with respective antibodies. Visualization of actin, microtubules, and GEF-H1 was performed by confocal microscopy. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Cntrl group.

Article Snippet: Rabbit antibodies against β-actin, GAPDH, and Myc were purchased from HuaBio-Antibodies; rabbit antibodies against RhoA and GEF-H1 were purchased from Cell Signaling Technology; mouse antibodies against TBC1D3C were purchased from Santa Cruz Technology; antibodies against Tctex, DIC, and 14-3-3 were purchased from Proteintech; mouse antibody against α-tubulin was purchased from Life Technologies; goat anti-rabbit and goat anti-mouse IgG-conjugated horseradish peroxidase antibodies were purchased from Promega; and Alexa Fluor–conjugated goat anti-mouse and rabbit IgG antibodies were bought from Jackson ImmunoResearch.

Techniques: Staining, Transfection, Labeling, Binding Assay, Immunoprecipitation, Confocal Microscopy

Journal: Life Science Alliance

Article Title: Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation

doi: 10.26508/lsa.202402585

Figure Lengend Snippet: (A) TBC1D3C interacts with GEF-H1 directly. GST or GST-TBC1D3C on glutathione Sepharose beads was incubated with MBP or MBP-GEF-H1 for pulldown assay. Pulled-down proteins were immunoblotted with MBP or GST antibodies. (B, C) TBC domain (residues 1–353) of TBC1D3C mediates its GEF-H1 interaction. HEK293T cells were co-transfected with full-length (FL) TBC1D3C or its truncation (B) or internal deletion mutants (C) and Myc-GEF-H1 . Immunoprecipitation was done with anti-FLAG antibody–conjugated agarose M2 beads, followed by immunoblotting with FLAG and Myc antibodies. (D) Residues R107 and D148 of TBC1D3C mediate its interaction with GEF-H1. FLAG-TBC1D3C wide type (WT) or its mutants R107A, D148A, or RD2A were co-expressed with Myc-GEF-H1 in HEK293T cells followed by immunoprecipitation and immunoblotting as indicated. (E, F) Dbl-homology (DH) and pleckstrin-homology (PH) domains of GEF-H1 interact with TBC1D3C. (E) Schematic of GEF-H1 and TBC1D3C domain structures. (F) FLAG-TBC1D3C was co-expressed with Myc-GEF-H1 or its deletion mutants in HEK293T cells, followed by immunoprecipitation and immunoblotting as indicated.

Article Snippet: Rabbit antibodies against β-actin, GAPDH, and Myc were purchased from HuaBio-Antibodies; rabbit antibodies against RhoA and GEF-H1 were purchased from Cell Signaling Technology; mouse antibodies against TBC1D3C were purchased from Santa Cruz Technology; antibodies against Tctex, DIC, and 14-3-3 were purchased from Proteintech; mouse antibody against α-tubulin was purchased from Life Technologies; goat anti-rabbit and goat anti-mouse IgG-conjugated horseradish peroxidase antibodies were purchased from Promega; and Alexa Fluor–conjugated goat anti-mouse and rabbit IgG antibodies were bought from Jackson ImmunoResearch.

Techniques: Incubation, Transfection, Immunoprecipitation, Western Blot

Journal: Life Science Alliance

Article Title: Decoupling actin assembly from microtubule disassembly by TBC1D3C-mediated direct GEF-H1 activation

doi: 10.26508/lsa.202402585

Figure Lengend Snippet: (A, B, C) Structured illumination microscopy imaging showing GEF-H1 relocalization from microtubules into the cytosol and actin stress fiber formation without microtubule disassembly; scale bar, 5 μm. (B) Correlation coefficients of GEF-H1 and microtubules in (A). (C) Quantification of F-actin in (A). TBC1D3C U2OS TetOn cells with or without Dox treatment were transfected with GFP-GEF-H1 , fixed, and stained with phalloidin and anti-tubulin antibodies. Images were captured by structured illumination microscopy. (D, E) Time-lapse live-cell imaging of microtubule, actin, and GEF-H1 dynamics after TBC1D3C induction. (E) Quantification of F-actin in (D). Cells were labeled with SiR-actin for actin (red) and transfected with GFP-GEF-H1 (green) and mCherry– tubulin (magenta); scale bar, 20 μm. (F) TBC1D3C expression leads to GEF-H1 accumulation in the cytoplasm. Lysates from TBC1D3C- or TBC1D3C-RD2A–expressing cells were subjected to centrifugation. GEF-H1 and microtubules in the supernatant and pellet were determined by Western blotting with respective antibodies. (G) Proposed model for TBC1D3C-directed GEF-H1 activation and decoupling of actin filament assembly and microtubule disassembly. GEF-H1 is tethered to polymerized microtubules by the dynein motor light-chain Tctex-1. TBC1D3C interacts directly with GEF-H1 to promote its release from microtubules to the cytosol without microtubule disassembly, leading to RhoA activation and subsequent actin filament assembly. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Cntrl group.

Article Snippet: Rabbit antibodies against β-actin, GAPDH, and Myc were purchased from HuaBio-Antibodies; rabbit antibodies against RhoA and GEF-H1 were purchased from Cell Signaling Technology; mouse antibodies against TBC1D3C were purchased from Santa Cruz Technology; antibodies against Tctex, DIC, and 14-3-3 were purchased from Proteintech; mouse antibody against α-tubulin was purchased from Life Technologies; goat anti-rabbit and goat anti-mouse IgG-conjugated horseradish peroxidase antibodies were purchased from Promega; and Alexa Fluor–conjugated goat anti-mouse and rabbit IgG antibodies were bought from Jackson ImmunoResearch.

Techniques: Microscopy, Imaging, Transfection, Staining, Live Cell Imaging, Labeling, Expressing, Centrifugation, Western Blot, Activation Assay