rabbit anti git1  (Proteintech)

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. In vitro phosphorylation of brain homogenate by recombinant active JNK1 revealed phosphorylation of GIT1 on S371 and S692. Mass spectrometry-identified sequences are shown for GIT1 (accession NP_001078923.1). Β. Representative autoradiograph and Coomassie Brilliant Blue (CBB) stained gel image are shown for recombinant GST-GIT1- wild-type (WT), -S371A, -S692A or S371A/S692A (SAA) variants after phosphorylation by GST-JNK1. JNK1 phosphorylated GIT1-WT and GIT1–S692A but not GIT1-S371A, indicating that S371 is the preferred JNK1 phosphorylation site. Phosphorylation of GIT1 by JNK1 was prevented by JNK inhibitor 10 µM SP600125. C. Quantitative data from 4 repeats of experiment shown in B . D. Domain map of human GIT1 (UniProt identifier: Q9Y2X7-3) shows predicted JNK1 phosphorylation sites (yellow circles). Abbreviations: Arf GTPase activating protein domain (GAP), 3x Ankyrin repeats (ANK), Spa-Homology domain (SHD), Synapse Localization Domain (SLD) and Paxillin Binding Domain (PBD). E. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in wild-type (WT) and Jnk1-/- whole brains. F. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in brains from DJNKI-1- infused adult mice. G. Active Arf1 was detected from pull downs using GST-GAT-GGA3 immobilized on glutathione beads. Cells expressed GIT1-variants together with Arf1-WT or constitutively active Arf1-Q71L. H. Quantitative data from 3 repeats of experiment described in G. Phosphorylation-site mutants of GIT1 did not alter Arf1 activity. Error bars represent standard error of the mean (SEM) and P-values were determined using Student’s t test.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: In Vitro, Recombinant, Mass Spectrometry, Autoradiography, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (25x optical slices, z=0.173 µm) depict EGFP-GIT1-WT and mRub-Lifeact in 16-DIV hippocampal neurons. Regions of interest (R.O.I.s; circles) depict where in the dendrite, spine and background mean intensity signals were acquired. B, D. Relative enrichment of EGFP-GIT1 variants in spines or dendrites, was calculated as a ratio of the mean intensity from the spine R.O.I. to the mean intensity from the R.O.I in the dendrite, after background subtraction, for the given treatments Jnk1-/- or TAT/DJNKI-1 (20 µM), 8 hours. C, E. Relative enrichment of mRuby-Lifeact fluorescence in spines was measured as for B and E. Mean data +/- S.E.Ms from a total of ∼80 spines from several neurons per condition are shown. Neurons originated from at least two independent mouse litters. P-values were calculated from Student’s two-tailed t test. F, G. We next checked the overall effect of DJNKI-1 inhibitor or Jnk1 knockout on GABAAR expression at the cell surface in the dendritic compartment. Both conditions increased general surface expression of GABAAR. H. In order to test the requirement for GIT1 in GABAAR trafficking, we characterised shRNA targeting GIT1. I. Representative maximum projection images of GABAAR β3 staining from 25 x optical sections (z=0.173 μm) are shown for WT and Jnk1-/- hippocampal neurons at 16 DIV. Staining was performed in non-permeabilized cells expressing mRuby, with non-targeting (NT)-shRNA or GIT1-shRNA and EGFP-GIT1 variants, as shown. White outlines were traced from mRuby fluorescence. Surface staining of GABAAR β3 is shown in pseudocolour. Representative images of total GABAAR β3 staining from an independent set of permeabilized cells is shown for comparison. J, K . Quantitative data from these experiments is shown for spines and dendrites. Mean data +/- S.E.M are shown from ∼70 dendritic spines per condition, in cells from at least two independent mouse litters. L, M. Cell surface expression of GABAAR β3 was measured from biotinylated samples. Representative blots show GABAAR β3 of lysates from before streptavidin-biotin enrichment (total) or after streptavidin-biotin enrichment (surface) are shown. Mean data from 3 repeats +/- S.E.M are shown. P-values were calculated from Student’s two-tailed t test.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Fluorescence, Two Tailed Test, Knock-Out, Expressing, shRNA, Staining, Comparison

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (32 x optical sections of 0.173 μm) of neurons expressing mRuby to visualize morphology. Neurons at 16 DIV were immunostained for endogenous GIT1 (green) and paxillin (PXN), 14-3-3ζ or β-PIX (magenta). B. Quantitative data show mean intensities of GIT1, PXN, 14-3-3ζ and β-PIX in spines. DJNKI- 1 increases GIT1 in dendritic spines compared to control (TAT), and reduces β-PIX in the same cell compartment. C. Mean intensity ratios of GIT1, PXN, 14-3-3ζ and β-PIX in dendritic spine/shaft. Mean data +/- SEMs from ∼42 spines per condition.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. We examined whether GABAAR β3, paxillin, β-PIX or 14-3-3ζ co-immunoprecipitated with GIT1 using cortex homogenates from WT and Jnk1-/- adult mice. +/- denotes addition or not of antibody. Representative immunoblots of input and GIT1 immunoprecipitates probed with indicated antibodies are shown. B. Quantitative data show the extent of GABAAR β3, PXN, β-PIX and 14-3-3ζ co-purification with GIT1 from brain. For 14-3-3ζ quantification, the background bands present in samples without GIT1 antibody were subtracted from bands in samples with GIT1 antibody. There was increased interaction between GIT1 and GABAAR β3, PXN and 14-3-3ζ in Jnk1-/- brain compared to WT. In contrast, interaction between GIT1 and β-PIX decreased. Histogram bars represent mean data from 4 experimental repeats +/- SEM. C. The effect of TAT or DJNKI-1 on GIT1 interaction with binding partners was determined. Cortical neurons at 16 DIV were treated with 20 µM TAT or DJNKI-1 for 8 hours. Representative immunoblots of GIT1 co-precipitations are shown. D. Band quantification from blots in . DJNKI-1-treated neurons show increased interaction between GIT1 and GABAAR β3, paxillin and 14-3-3ζ, whereas β-PIX interaction with GIT1 reduced. Bars show mean data from 5 repeats ± SEM. E. We tested whether GIT1-S371 phosphorylation site mutants altered these interactions. HEK-293 cells were transfected with EGFP-GIT1-WT, EGFP-GIT1-S371A or EGFP-GIT1-S371D and either Flag (as a control), Flag-PXN-WT or Flag-PXN-S178A, as indicated. Representative blots of input and co-immunoprecipitations are shown. F. Quantitative band intensities from E are shown using log10 scale. PXN-S178A interacted most highly with GIT1-S371A. Mean data ± SEM from 4 repeats are shown. Student’s t-test p-values above bars are compared to samples with EGFP-GTI1-WT and Flag-PXN-WT. G. The same approach was used to test endogenous 14-3-3ζ and Flag-β-PIX interaction with GIT1. Representative blots are shown. H. Quantitative data for Flag-β−PIX interaction with EGFP-GIT1 variants. I. Quantitative data for 14-3-3ζ interaction with EGFP-GIT1 variants. Endogenous 14-3-3ζ interacts most highly with EGFP-GIT1-S371A. Bars ± SEM represent means from 3 repeats. p-values were calculated from Student’s two-tailed t-test and are compared to conditions where WT is expressed. IB: immunoblot, IP: immunoprecipitation.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Immunoprecipitation, Western Blot, Copurification, Binding Assay, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (22 x optical sections of 0.173 μm) showing staining of GABAAR β3 in non-permeabilised 16-DIV hippocampal neurons (magenta). Cells co-express mRuby with NT-shRNA or GIT1shRNA, as indicated. Cell tracing was based on mRuby images. Cells were treated with 10 µM R18 for 24 h and 20 µM TAT or DJNKI-1 for 8 h. B. Quantification of GABAAR β3 enrichment in dendritic spines. R18 prevented DJNKI-1-induced GABAAR β3 increase at the dendritic spines. C. R18 prevented GIT1-S371A-induced increase in GABAAR β3 at the cell surface. Histogram bars represent means from ∼51 spines per condition from several neurons. P-values were calculated from Student’s two-tailed t-tests were compared to control (TAT) unless otherwise indicated. D. Neurons were co-transfected at 7 DIV with EGFP-Dynamin 2-WT or EGFP-Dynamin 2- K44A and treated with 20 µM TAT or DJNKI-1 for 8 h. Representative images are shown. E. Mean intensities for GABAAR β3 are shown from non-permeabilised cells in mushroom spines. GABAAR surface expression increased in DJNKI-1-treated neurons, even when the K44A mutant is expressed. Mean data +/- SEMs from ∼70 spines per condition are shown. p- values (calculated from Student’s two-tailed t test) were compared to control (TAT) unless otherwise indicated by brackets.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Staining, shRNA, Two Tailed Test, Control, Transfection, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: We propose that when JNK1 active it phosphorylates GIT1 on S371. This reduces levels of GABAAR at the cell surface, both at excitatory synapses and at extrasynapatic sites. Conversely when JNK is inhibited or Jnk1 genetically deleted, GIT1 associates with a paxillin and 14-33-containing receptor complex at the membrane. GIT1 phosphorylation by JNK on S371 dissociates it from binding to this complex, leading to run-down of receptor surface expression. The consequence for neuronal physiology is increased inhibitor post synaptic currents and increased tonic inhibitory current.

Article Snippet: For immunoblotting dilutions were as follows: 1:1000 paxillin (Bioscience), 1:500 GABAβ3 (Neuromab), 1:2000 GIT1 (Santa-Cruz), 1:1000 β-PIX (Bioscience), 1:1000 14-3-3ζ (Santa-Cruz), 1:2000 FLAG (Sigma Aldrich), 1:10,000 EGFP (JL-8, Clontech), 1: 2000 HA (Cell Signaling), 1:1000 JNK1 (Bioscience), 1:50,000 anti-rabbit and anti-mouse secondary HRP antibodies (Millipore).

Techniques: Membrane, Binding Assay, Expressing

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. In vitro phosphorylation of brain homogenate by recombinant active JNK1 revealed phosphorylation of GIT1 on S371 and S692. Mass spectrometry-identified sequences are shown for GIT1 (accession NP_001078923.1). Β. Representative autoradiograph and Coomassie Brilliant Blue (CBB) stained gel image are shown for recombinant GST-GIT1- wild-type (WT), -S371A, -S692A or S371A/S692A (SAA) variants after phosphorylation by GST-JNK1. JNK1 phosphorylated GIT1-WT and GIT1–S692A but not GIT1-S371A, indicating that S371 is the preferred JNK1 phosphorylation site. Phosphorylation of GIT1 by JNK1 was prevented by JNK inhibitor 10 µM SP600125. C. Quantitative data from 4 repeats of experiment shown in B . D. Domain map of human GIT1 (UniProt identifier: Q9Y2X7-3) shows predicted JNK1 phosphorylation sites (yellow circles). Abbreviations: Arf GTPase activating protein domain (GAP), 3x Ankyrin repeats (ANK), Spa-Homology domain (SHD), Synapse Localization Domain (SLD) and Paxillin Binding Domain (PBD). E. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in wild-type (WT) and Jnk1-/- whole brains. F. Quantitative LC-MS/MS data of GIT1-S371 phosphorylation in brains from DJNKI-1- infused adult mice. G. Active Arf1 was detected from pull downs using GST-GAT-GGA3 immobilized on glutathione beads. Cells expressed GIT1-variants together with Arf1-WT or constitutively active Arf1-Q71L. H. Quantitative data from 3 repeats of experiment described in G. Phosphorylation-site mutants of GIT1 did not alter Arf1 activity. Error bars represent standard error of the mean (SEM) and P-values were determined using Student’s t test.

Article Snippet: pEGFP-C1 GIT1 (EGFP GIT1 WT) (Addgene plasmid: # 15226) and Flag-beta PIXa (Addgene plasmid: # 15234) were gifts from Rick Horwitz and pSuper-GIT1 shRNA was from Huaye Zhang.

Techniques: In Vitro, Recombinant, Mass Spectrometry, Autoradiography, Staining, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Activity Assay

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (25x optical slices, z=0.173 µm) depict EGFP-GIT1-WT and mRub-Lifeact in 16-DIV hippocampal neurons. Regions of interest (R.O.I.s; circles) depict where in the dendrite, spine and background mean intensity signals were acquired. B, D. Relative enrichment of EGFP-GIT1 variants in spines or dendrites, was calculated as a ratio of the mean intensity from the spine R.O.I. to the mean intensity from the R.O.I in the dendrite, after background subtraction, for the given treatments Jnk1-/- or TAT/DJNKI-1 (20 µM), 8 hours. C, E. Relative enrichment of mRuby-Lifeact fluorescence in spines was measured as for B and E. Mean data +/- S.E.Ms from a total of ∼80 spines from several neurons per condition are shown. Neurons originated from at least two independent mouse litters. P-values were calculated from Student’s two-tailed t test. F, G. We next checked the overall effect of DJNKI-1 inhibitor or Jnk1 knockout on GABAAR expression at the cell surface in the dendritic compartment. Both conditions increased general surface expression of GABAAR. H. In order to test the requirement for GIT1 in GABAAR trafficking, we characterised shRNA targeting GIT1. I. Representative maximum projection images of GABAAR β3 staining from 25 x optical sections (z=0.173 μm) are shown for WT and Jnk1-/- hippocampal neurons at 16 DIV. Staining was performed in non-permeabilized cells expressing mRuby, with non-targeting (NT)-shRNA or GIT1-shRNA and EGFP-GIT1 variants, as shown. White outlines were traced from mRuby fluorescence. Surface staining of GABAAR β3 is shown in pseudocolour. Representative images of total GABAAR β3 staining from an independent set of permeabilized cells is shown for comparison. J, K . Quantitative data from these experiments is shown for spines and dendrites. Mean data +/- S.E.M are shown from ∼70 dendritic spines per condition, in cells from at least two independent mouse litters. L, M. Cell surface expression of GABAAR β3 was measured from biotinylated samples. Representative blots show GABAAR β3 of lysates from before streptavidin-biotin enrichment (total) or after streptavidin-biotin enrichment (surface) are shown. Mean data from 3 repeats +/- S.E.M are shown. P-values were calculated from Student’s two-tailed t test.

Article Snippet: pEGFP-C1 GIT1 (EGFP GIT1 WT) (Addgene plasmid: # 15226) and Flag-beta PIXa (Addgene plasmid: # 15234) were gifts from Rick Horwitz and pSuper-GIT1 shRNA was from Huaye Zhang.

Techniques: Fluorescence, Two Tailed Test, Knock-Out, Expressing, shRNA, Staining, Comparison

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (32 x optical sections of 0.173 μm) of neurons expressing mRuby to visualize morphology. Neurons at 16 DIV were immunostained for endogenous GIT1 (green) and paxillin (PXN), 14-3-3ζ or β-PIX (magenta). B. Quantitative data show mean intensities of GIT1, PXN, 14-3-3ζ and β-PIX in spines. DJNKI- 1 increases GIT1 in dendritic spines compared to control (TAT), and reduces β-PIX in the same cell compartment. C. Mean intensity ratios of GIT1, PXN, 14-3-3ζ and β-PIX in dendritic spine/shaft. Mean data +/- SEMs from ∼42 spines per condition.

Article Snippet: pEGFP-C1 GIT1 (EGFP GIT1 WT) (Addgene plasmid: # 15226) and Flag-beta PIXa (Addgene plasmid: # 15234) were gifts from Rick Horwitz and pSuper-GIT1 shRNA was from Huaye Zhang.

Techniques: Expressing, Control

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. We examined whether GABAAR β3, paxillin, β-PIX or 14-3-3ζ co-immunoprecipitated with GIT1 using cortex homogenates from WT and Jnk1-/- adult mice. +/- denotes addition or not of antibody. Representative immunoblots of input and GIT1 immunoprecipitates probed with indicated antibodies are shown. B. Quantitative data show the extent of GABAAR β3, PXN, β-PIX and 14-3-3ζ co-purification with GIT1 from brain. For 14-3-3ζ quantification, the background bands present in samples without GIT1 antibody were subtracted from bands in samples with GIT1 antibody. There was increased interaction between GIT1 and GABAAR β3, PXN and 14-3-3ζ in Jnk1-/- brain compared to WT. In contrast, interaction between GIT1 and β-PIX decreased. Histogram bars represent mean data from 4 experimental repeats +/- SEM. C. The effect of TAT or DJNKI-1 on GIT1 interaction with binding partners was determined. Cortical neurons at 16 DIV were treated with 20 µM TAT or DJNKI-1 for 8 hours. Representative immunoblots of GIT1 co-precipitations are shown. D. Band quantification from blots in . DJNKI-1-treated neurons show increased interaction between GIT1 and GABAAR β3, paxillin and 14-3-3ζ, whereas β-PIX interaction with GIT1 reduced. Bars show mean data from 5 repeats ± SEM. E. We tested whether GIT1-S371 phosphorylation site mutants altered these interactions. HEK-293 cells were transfected with EGFP-GIT1-WT, EGFP-GIT1-S371A or EGFP-GIT1-S371D and either Flag (as a control), Flag-PXN-WT or Flag-PXN-S178A, as indicated. Representative blots of input and co-immunoprecipitations are shown. F. Quantitative band intensities from E are shown using log10 scale. PXN-S178A interacted most highly with GIT1-S371A. Mean data ± SEM from 4 repeats are shown. Student’s t-test p-values above bars are compared to samples with EGFP-GTI1-WT and Flag-PXN-WT. G. The same approach was used to test endogenous 14-3-3ζ and Flag-β-PIX interaction with GIT1. Representative blots are shown. H. Quantitative data for Flag-β−PIX interaction with EGFP-GIT1 variants. I. Quantitative data for 14-3-3ζ interaction with EGFP-GIT1 variants. Endogenous 14-3-3ζ interacts most highly with EGFP-GIT1-S371A. Bars ± SEM represent means from 3 repeats. p-values were calculated from Student’s two-tailed t-test and are compared to conditions where WT is expressed. IB: immunoblot, IP: immunoprecipitation.

Article Snippet: pEGFP-C1 GIT1 (EGFP GIT1 WT) (Addgene plasmid: # 15226) and Flag-beta PIXa (Addgene plasmid: # 15234) were gifts from Rick Horwitz and pSuper-GIT1 shRNA was from Huaye Zhang.

Techniques: Immunoprecipitation, Western Blot, Copurification, Binding Assay, Transfection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: A. Representative maximum projection images (22 x optical sections of 0.173 μm) showing staining of GABAAR β3 in non-permeabilised 16-DIV hippocampal neurons (magenta). Cells co-express mRuby with NT-shRNA or GIT1shRNA, as indicated. Cell tracing was based on mRuby images. Cells were treated with 10 µM R18 for 24 h and 20 µM TAT or DJNKI-1 for 8 h. B. Quantification of GABAAR β3 enrichment in dendritic spines. R18 prevented DJNKI-1-induced GABAAR β3 increase at the dendritic spines. C. R18 prevented GIT1-S371A-induced increase in GABAAR β3 at the cell surface. Histogram bars represent means from ∼51 spines per condition from several neurons. P-values were calculated from Student’s two-tailed t-tests were compared to control (TAT) unless otherwise indicated. D. Neurons were co-transfected at 7 DIV with EGFP-Dynamin 2-WT or EGFP-Dynamin 2- K44A and treated with 20 µM TAT or DJNKI-1 for 8 h. Representative images are shown. E. Mean intensities for GABAAR β3 are shown from non-permeabilised cells in mushroom spines. GABAAR surface expression increased in DJNKI-1-treated neurons, even when the K44A mutant is expressed. Mean data +/- SEMs from ∼70 spines per condition are shown. p- values (calculated from Student’s two-tailed t test) were compared to control (TAT) unless otherwise indicated by brackets.

Article Snippet: pEGFP-C1 GIT1 (EGFP GIT1 WT) (Addgene plasmid: # 15226) and Flag-beta PIXa (Addgene plasmid: # 15234) were gifts from Rick Horwitz and pSuper-GIT1 shRNA was from Huaye Zhang.

Techniques: Staining, shRNA, Two Tailed Test, Control, Transfection, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: JNK regulates GABAAR expression at the cell surface via the receptor clustering protein GIT1 (ArfGAP1)

doi: 10.1101/2025.01.22.634236

Figure Lengend Snippet: We propose that when JNK1 active it phosphorylates GIT1 on S371. This reduces levels of GABAAR at the cell surface, both at excitatory synapses and at extrasynapatic sites. Conversely when JNK is inhibited or Jnk1 genetically deleted, GIT1 associates with a paxillin and 14-33-containing receptor complex at the membrane. GIT1 phosphorylation by JNK on S371 dissociates it from binding to this complex, leading to run-down of receptor surface expression. The consequence for neuronal physiology is increased inhibitor post synaptic currents and increased tonic inhibitory current.

Article Snippet: pEGFP-C1 GIT1 (EGFP GIT1 WT) (Addgene plasmid: # 15226) and Flag-beta PIXa (Addgene plasmid: # 15234) were gifts from Rick Horwitz and pSuper-GIT1 shRNA was from Huaye Zhang.

Techniques: Membrane, Binding Assay, Expressing

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) Relative Git1 intensity in the Smo-TurboID proteomic results. (B) Git1 is biotinylated by Smo-TurboID independent of Shh treatment. Smo-V5-TurboID stable cells were infected with lentiviruses expressing YFP-Git1. Cells were treated with Shh for 1h, and cell lysates were used for purification with Streptavidin beads. a-Tubulin is used as loading control. (C) Git1 localizes to the basal body. NIH3T3 cells were transfected with YFP-Git1, and stained for primary cilium (Arl13b, red), basal body (Pericentrin, magenta), and nucleus (DAPI, blue). In addition to its putative distribution in the cytosol, Git1 also localizes to the basal body. Scale bar, 5 μm, 2 μm (inset). (D, F) Immunofluorescence imaging of Smo and phosphorylated Smo (pSmo) in WT and Git1-null cells. The cells are treated with 1 μg/ml recombinant Shh or vehicle. Primary cilium is highlighted by acetylated Tubulin (red). Scale bar, 5 μm, 2 μm (inset). (E, G) Quantification of ciliary Smo and pSmo immunofluorescence intensity (AU). n = 100-150 cells/condition from three biological replicates. (H) Representative images of immunofluorescence staining in Smo-TurboID cells transfected with control shRNA or shRNA against Git1. Cells were fixed 72hr after lentiviral infection and stained with PKA-C (green), Arl13b (red) and nucleus (DAPI, blue). Scale bar, 5 μm, 2 μm (inset). (I) Left: Quantification of ciliary PKA-C immunofluorescence intensity (AU), n = 90 cells/condition from three biological replicates; Right: Quantification of % ciliary PKA-C relative to total nucleus in the field. n= 15 fields per condition. Statistics in E, G, I (Left): two-way ANOVA followed by Tukey’s multiple comparison test. Statistics in I (Right) is assessed by one-way ANOVA followed by Sidak’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

Techniques: Infection, Expressing, Purification, Control, Transfection, Staining, Immunofluorescence, Imaging, Recombinant, shRNA, Comparison

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) guide RNA was designed to target exon 2 of mouse Git1 . (B) The gRNA targeting region in mouse genomic was amplified by genomic PCR, ligated into TOPO vector, and transfected into chemically competent cells. 20 bacterial colonies of each cell clones were randomly picked and sequenced. The Sanger sequencing results were aligned with the genome sequence of the M. musculus. Single base pair deletion and insertion are identified, resulting in biallelic frameshift or early termination. (C) Immunoblot of Git1-null cell lines showing that Git1 protein is not detected in knockout cells. GAPDH is used as the loading control. (D) Cilium staining in WT and Git1-null cell colonies. Primary cilium is marked with Arl13b (red). Scale bar, 10 μm. (E) Quantification of cilium length, n = 60 cells/condition from 3 biological replicates. (F) Git1 transcript levels in cells transfected with shRNA against Git1. Statistics in (E-F): one-way ANOVA followed by Sidak’s multiple comparisons test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

Techniques: Amplification, Plasmid Preparation, Transfection, Clone Assay, Sequencing, Western Blot, Knock-Out, Control, Staining, shRNA

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) Immunofluorescence image of Grk2-V5 (red) and YFP-Git1 (green) in NIH3T3. γTub labels centrosome (magenta). Scale bars, 5 μm and 2 μm (inset). (B) Co-immunoprecipitation blot showing interaction between Git1-Flag and Grk2-V5 when co-expressed in 293T cells. (C) Representative images of Grk2 levels at the basal body and in the cilium after Shh stimulation in WT and Git1-null cells. (D, E) Quantification of basal body and ciliary Grk2 intensity in WT and Git1-null cells. n=90 cells/condition from three biological replicates. Data are shown as mean ± SD. Statistics in D and E: two-way ANOVA followed by Tukey’s multiple comparison test. ** or ## p < 0.001, ****p < 0.0001, versus time 0.

Article Snippet: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

Techniques: Immunofluorescence, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) qPCR measurement of Gli1 transcript levels in wild-type NIH3T3 (WT) and Git1-null cell colonies. Cells were stimulated with Shh or vehicle for 24h. (B) Immunoblot of Gli3, Gli1 in WT and Git1-null cell colonies. a-Tubulin (aTub) is used as the loading control. (C and D) Quantification of immunoblot intensity of Gli3 and Gli1. n =4 independent experiments. (E) Immunofluorescent imaging of WT and Git1-null cells with or without Shh treatment. Cells were stained for Gli2 (green), and primary cilium (acTub, red). Scale bars, 5 μm and 2 μm (inset). (F) Quantification of Gli2 signal at the ciliary tip in WT and Git1-null cells. n=150 cells from 3 biological replicates. (G) Representative images of Git1-null cells infected with lentiviruses expressing Grk2-V5-Δ1Arl13b. (H) qPCR measurement of Gli1 transcript levels in WT and Git1-null cell colonies that express the indicated construct. The control plasmid refers to V5-Δ1Arl13b backbone. Statistics in A, C, D, F, H: two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

Techniques: Western Blot, Control, Imaging, Staining, Infection, Expressing, Construct, Plasmid Preparation, Comparison

Journal: bioRxiv

Article Title: Proximity based proteomics reveals Git1 as a regulator of Smoothened signaling

doi: 10.1101/2025.01.06.631593

Figure Lengend Snippet: (A) Experimental workflow of GNP primary culture and treatment. (B) qPCR measurement of Git1 transcript levels in GNPs at the end of the experiment. (C) Hh signaling intensity is assessed by Gli1 transcript levels in GNPs at the end of the experiment. (D) Immunostaining of Edu (magenta) incorporation in primary cultured GNPs. Cells are infected with lentivirus expressing control or Git1 shRNA. (E) quantification of Edu incorporation into the GNP nucleus. n = 10 fields for each condition. (F) Schematic view of Git1’s function in Hh signaling. Git1 at the ciliary base facilitates Grk2’s interaction with Smo; it also promotes Grk2 translocation into the cilium to effectively phosphorylate Smo. Without Git1, Grk2 fails to phosphorylate Smo, leading to reduced Hh signaling. Data in B, C, E are shown as Mean ± SD. Statistics: two-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Article Snippet: To observe localization and activity of Git1, YFP was fused to the N terminus of human Git1 (Addgene, 15225) and cloned into FUGW backbone (Addgene, 14883).

Techniques: Immunostaining, Cell Culture, Infection, Expressing, Control, shRNA, Translocation Assay, Comparison