Journal: iScience

Article Title: ILF3 prion-like domain regulates gene expression and fear memory under chronic stress

doi: 10.1016/j.isci.2023.106229

Figure Lengend Snippet:

Article Snippet: Ribosome-bound RNA was isolated using a Direct-zol RNA MicroPrep Kit (Zymo Research, Irvine, CA).

Techniques: Recombinant, RNA HS Assay, CRISPR, Sequencing, Control, Software, Battery

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 1. CDGI interacts with LRRK2. (A) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (B) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from LRRK2 WT and KO mouse whole brains were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (C) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from CDGI WT and KO mouse whole brains were subjected to IP with anti-CDGI followed by anti-LRRK2 and anti-CDGI immunoblotting. (D) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse striatal lysates. Lysates prepared from LRRK2 WT and KO mouse stria- tum were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (E) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged F1 (the GEF domain), F2 (the EF-hands domain), F3 (the DAG domain), or full-length (FL) CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was fol- lowed by anti-flag or anti-MYC immunoblotting. A schematic representation of CDGI F1, F2, and F3 domains is shown. (F) Co-IP analysis of the interaction between V5- tagged CDGI and flag-tagged LRRK2 fragments in cotransfected HEK 293T cells. Co-IP with anti-V5 was followed by anti-flag or anti-V5 immunoblotting. A schematic representation of the different LRRK2 fragments is shown. (G) Co-IP analysis of the interaction between V5-tagged CDGI and MYC-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-V5 immunoblotting. (H) Co-IP analysis of the interaction between flag-tagged CDGI-F1 domain and Myc-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (I) Coimmunostaining of CDGI (green), LRRK2 (red), and DARPP32 (magenta) in mouse striatal sections. aa, amino acid.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 2. CDGI acts as a potential GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release. (A) GTP loading analysis of LRRK2 by an in vivo 32P- orthophosphate labeling in HEK 293T cells overexpressing the indicated plasmids. The migration of GTP and GDP is indicated. The Ca2+ ionophore A23187 and TPA were applied. (B) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (C) GTP loading analysis of LRRK2 R1441C (RC) and G2019S (GS) with CDG-WT or GW by an in vivo 32P-orthophosphate labeling in HEK 293T cells. TN (T1348N) was a negative control. (D) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (E) The levels of LRRK2 bound to GTP were analyzed by pull-down assays with GTP-agarose from HEK 293T cells. (F) Quantification of LRRK2 bound to GTP-agarose beads. Data were normalized to LRRK2-WT alone in (B), (D), and (F). (G) GTP loading analysis of LRRK2 by an in vivo 32P-orthophosphate labeling in WT stria- tal neurons compared to CDGI KO neurons transduced by LV-CDGI-WT or LV-CDGI-GW. (H) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). Data were normalized to WT neurons. (I) A diagram of the guanine nucleotide exchange assay is illustrated. Free BODIPY-FL-GDP is quenched with a low fluorescence in the solutions while showing increased fluorescence upon binding to GTPases. GEF catalyzes the exchange of preloaded BODIPY-FL-GDP for GTP causing a decrease in fluorescence. (J) Recombinant LRRK2 preloaded with BODIPY-FL-GDP was incubated with recombinant GST-CDGI proteins in the presence of excess cold GTP. Nucleotide exchange on LRRK2 was monitored by the fluorescence intensity change with different concentrations of CDGI-WT every 36 s for 15 min. Data are the means ± SEM, n = 3. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not statistically significant.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Binding Assay, Activity Assay, In Vivo, Labeling, Migration, Negative Control, Fluorescence, Recombinant, Incubation

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 3. CDGI increases LRRK2 membrane association. (A to C) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in HEK 293T cells co- transfected with LRRK2 and CDGI-WT or GW. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Samples were immunoblotted with anti-MYC, V5, LAMP1, Rab10, and phosphor-Rab10. Data were normalized to LRRK2-MYC alone. (D and E) Liquid nitrogen freeze-thaw methods assessing LRRK2 membrane association. HEK 293T cells with stably expressed eGFP-LRRK2 were transfected with or without mCherry-CDGI. After 48 hours, cells were permeabilized by liquid nitrogen freeze-thaw to deplete cytosol and then fixed, immunostained with LAMP1, and visualized by confocal microscopy. Scale bar, 20 μM. Data were normalized to eGFP-LRRK2 stable cells transfected with mCherry. (F and G) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in CDGI WT striatal neu- rons compared to CDGI KO neurons. CDGI WT and KO striatal neurons were treated with A23187 and TPA before being harvested and fractionated into cytosol and mem- brane fractions. Data were normalized to CDGI-WT neurons. Data are the means ± SEM, n = 3. One-way ANOVA followed by Tukey’s post hoc test was used for the data analysis of multiple comparisons, and Student’s t tests (unpaired, two-tailed) were used for the data analysis of two comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Membrane, Cell Fractionation, Transfection, Stable Transfection, Confocal Microscopy, Two Tailed Test

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 4. CDGI acts upstream of LRRK2 to regulate retinal neurodegeneration. (A) Representative images of eye morphology of 1-week-old flies of the indicated geno- types by light microscopy. Drosophila LRRK2 (dLRRK) RNAi knockdown (dLRRKRI), human LRRK2 WT (LRRK2WT), and LRRK2 mutant GS or RC (LRRK2GS or LRRK2RC) were coex- pressed with human CDGI WT (CDGIWT) or CDGI-GW (CDGIGW) in fly eyes by a GMR-GAL4 driver (GMR > CDGI/LRRK2). For each genotype, images were taken from at least 10 flies. Scale bar, 100 μm. (B) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by SEM. Scale bar, 100 μm. (C) The fly eye size was quantified by ImageJ for each genotype. n = 10. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Light Microscopy, Knockdown, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 5. CDGI acts upstream of LRRK2 to regulate striatal and DA neurodegeneration. (A) Representative images of NeuN staining of mouse striatum. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, the striatal neurons were immuno- labeled by a NeuN antibody. Scale bar, 250 μm. (B) Quantification of NeuN-positive neurons in the striatum using an unbiased stereological method with stereo investiga- tor software from five animals per group. (C) Representative images of DA neuron staining in mouse SNpc. After 9 to 10 months of viral injection, the DA neurons were immunolabeled by a TH antibody. Scale bar, 500 μm. (D) Quantification of TH-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from five to seven animals per group. Data are mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, ** P < 0.01, and ***P < 0.001.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Staining, Injection, Labeling, Software, Immunolabeling

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 6. CDGI acts upstream of LRRK2 to regulate locomotor behavioral deficits. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, a battery of behavioral tests was performed. (A) Open-field test. The total time spent at the center was analyzed. (B) Open-field test. The total time spent at the corner was analyzed. (C) Rotarod test. The average retention time was analyzed. (D) Pole test to monitor behav- ioral abnormalities. The total time to descend to the bottom was recorded. Data are mean ± SEM, n = 5 to 9 per group, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Injection, Battery

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 7. Model of CDGI regulation of LRRK2 function and LRRK2-induced neurodegeneration. LRRK2 GTPase cycle is between inactive off GDP-bound state and active on GTP-bound state. CDGI binds to LRRK2 serving as a physiological GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release, and membrane association and in turn regulates LRRK2-induced striatal and DA neurodegeneration and behavioral deficits.

Article Snippet: Rabbit anti- CDG1 (Graybiel Laboratory), rabbit anti- RasGRP2 (ab137608, Abcam), mouse monoclonal anti- LRRK2 (clone N138/6, University of California Davis/NIH NeuroMab facility), rabbit monoclonal anti- LRRK2 (D18E12, #13046, Cell Signaling Technology), rabbit monoclonal anti- LRRK2 (c41- 2, ab133474, Abcam), rabbit anti- TH polyclonal antibody (NB300- 109, Novus Biologicals), mouse anti- NeuN (ABN78, Millipore Sigma), rat anti- DARPP32 (MAB4230SP, R&D), mouse anti- EEA1 (68065- 1- Ig, Proteintech), sheep antiTGN46 (AHP500GT, Bio- Rad), rat anti- LAMP1 [D4B, Developmental Studies Hybridoma Bank (DSHB)], mouse monoclonal anti-LAMP1 (clone H4A3, DSHB), rabbit anti- LAMP1 (ab24170, Abcam), mouse anti- KDEL (NBP1- 97469, Novus), rat anti- Mac2 (Gal3) (125401, BioLegend), mouse monoclonal anti- V5 antibody (R96025, Thermo Fisher Scientific), mouse monoclonal anti- MYC (M4439, Sigma- Aldrich), mouse monoclonal anti–V5–horseradish peroxidase (HRP) (V2260, Sigma- Aldrich), mouse monoclonal antiFlag (F3165, Sigma- Aldrich), mouse monoclonal anti–Flag- HRP (A8592, Sigma- Aldrich), mouse monoclonal anti–hemagglutinin (HA; H3663, Sigma- Aldrich), mouse monoclonal anti–HA- HRP (H6533, Sigma- Aldrich), mouse monoclonal anti- ACTIN (A2066, Sigma- Aldrich), HRP- linked anti- rabbit immunoglobulin G (IgG; 111- 035- 144, Jackson ImmunoResearch), HRP- linked anti- mouse IgG (115- 035- 166, Jackson ImmunoResearch), Alexa Fluor 488 antimouse IgG (115- 545- 166, Jackson ImmunoResearch), Alexa Fluor 488 anti- rabbit IgG (111- 545- 144, Jackson ImmunoResearch), Alexa Fluor 405 anti- rat IgG (A48261, Thermo Fisher Scientific), Alexa Fluor 647 anti- rabbit IgG (A32733, Thermo Fisher Scientific), TPA (P8139, Sigma- Aldrich), A23187 (C7522, Sigma- Aldrich), LLOMe (16008, Cayman), Alexa Fluor 594 Tyramide Reagent (B40957, Thermo Fisher Scientific), BODIPY FL GDP (G22360, Thermo Fisher Scientific), GST tag recombinant human LRRK2 protein (PV4873, Thermo Fisher Scientific).

Techniques: Binding Assay, Activity Assay, Membrane

Journal: iScience

Article Title: ILF3 prion-like domain regulates gene expression and fear memory under chronic stress

doi: 10.1016/j.isci.2023.106229

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were the anti-ILF3 rabbit monoclonal antibody (Abcam), anti-TDP-43 rabbit polyclonal antibody (10782-2-AP, Proteintech, Rosemont, IL, USA), anti-hnRNPA1 rabbit monoclonal antibody (#8443, Cell Signaling Technology, Danvers, MA), and anti-c-Fos rabbit monoclonal antibody (#2250, Cell Signaling Technology).

Techniques: Recombinant, RNA HS Assay, CRISPR, Sequencing, Control, Software, Battery

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 1. CDGI interacts with LRRK2. (A) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (B) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from LRRK2 WT and KO mouse whole brains were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (C) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from CDGI WT and KO mouse whole brains were subjected to IP with anti-CDGI followed by anti-LRRK2 and anti-CDGI immunoblotting. (D) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse striatal lysates. Lysates prepared from LRRK2 WT and KO mouse stria- tum were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (E) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged F1 (the GEF domain), F2 (the EF-hands domain), F3 (the DAG domain), or full-length (FL) CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was fol- lowed by anti-flag or anti-MYC immunoblotting. A schematic representation of CDGI F1, F2, and F3 domains is shown. (F) Co-IP analysis of the interaction between V5- tagged CDGI and flag-tagged LRRK2 fragments in cotransfected HEK 293T cells. Co-IP with anti-V5 was followed by anti-flag or anti-V5 immunoblotting. A schematic representation of the different LRRK2 fragments is shown. (G) Co-IP analysis of the interaction between V5-tagged CDGI and MYC-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-V5 immunoblotting. (H) Co-IP analysis of the interaction between flag-tagged CDGI-F1 domain and Myc-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (I) Coimmunostaining of CDGI (green), LRRK2 (red), and DARPP32 (magenta) in mouse striatal sections. aa, amino acid.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 2. CDGI acts as a potential GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release. (A) GTP loading analysis of LRRK2 by an in vivo 32P- orthophosphate labeling in HEK 293T cells overexpressing the indicated plasmids. The migration of GTP and GDP is indicated. The Ca2+ ionophore A23187 and TPA were applied. (B) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (C) GTP loading analysis of LRRK2 R1441C (RC) and G2019S (GS) with CDG-WT or GW by an in vivo 32P-orthophosphate labeling in HEK 293T cells. TN (T1348N) was a negative control. (D) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (E) The levels of LRRK2 bound to GTP were analyzed by pull-down assays with GTP-agarose from HEK 293T cells. (F) Quantification of LRRK2 bound to GTP-agarose beads. Data were normalized to LRRK2-WT alone in (B), (D), and (F). (G) GTP loading analysis of LRRK2 by an in vivo 32P-orthophosphate labeling in WT stria- tal neurons compared to CDGI KO neurons transduced by LV-CDGI-WT or LV-CDGI-GW. (H) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). Data were normalized to WT neurons. (I) A diagram of the guanine nucleotide exchange assay is illustrated. Free BODIPY-FL-GDP is quenched with a low fluorescence in the solutions while showing increased fluorescence upon binding to GTPases. GEF catalyzes the exchange of preloaded BODIPY-FL-GDP for GTP causing a decrease in fluorescence. (J) Recombinant LRRK2 preloaded with BODIPY-FL-GDP was incubated with recombinant GST-CDGI proteins in the presence of excess cold GTP. Nucleotide exchange on LRRK2 was monitored by the fluorescence intensity change with different concentrations of CDGI-WT every 36 s for 15 min. Data are the means ± SEM, n = 3. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not statistically significant.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Binding Assay, Activity Assay, In Vivo, Labeling, Migration, Negative Control, Fluorescence, Recombinant, Incubation

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 3. CDGI increases LRRK2 membrane association. (A to C) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in HEK 293T cells co- transfected with LRRK2 and CDGI-WT or GW. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Samples were immunoblotted with anti-MYC, V5, LAMP1, Rab10, and phosphor-Rab10. Data were normalized to LRRK2-MYC alone. (D and E) Liquid nitrogen freeze-thaw methods assessing LRRK2 membrane association. HEK 293T cells with stably expressed eGFP-LRRK2 were transfected with or without mCherry-CDGI. After 48 hours, cells were permeabilized by liquid nitrogen freeze-thaw to deplete cytosol and then fixed, immunostained with LAMP1, and visualized by confocal microscopy. Scale bar, 20 μM. Data were normalized to eGFP-LRRK2 stable cells transfected with mCherry. (F and G) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in CDGI WT striatal neu- rons compared to CDGI KO neurons. CDGI WT and KO striatal neurons were treated with A23187 and TPA before being harvested and fractionated into cytosol and mem- brane fractions. Data were normalized to CDGI-WT neurons. Data are the means ± SEM, n = 3. One-way ANOVA followed by Tukey’s post hoc test was used for the data analysis of multiple comparisons, and Student’s t tests (unpaired, two-tailed) were used for the data analysis of two comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Membrane, Cell Fractionation, Transfection, Stable Transfection, Confocal Microscopy, Two Tailed Test

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 4. CDGI acts upstream of LRRK2 to regulate retinal neurodegeneration. (A) Representative images of eye morphology of 1-week-old flies of the indicated geno- types by light microscopy. Drosophila LRRK2 (dLRRK) RNAi knockdown (dLRRKRI), human LRRK2 WT (LRRK2WT), and LRRK2 mutant GS or RC (LRRK2GS or LRRK2RC) were coex- pressed with human CDGI WT (CDGIWT) or CDGI-GW (CDGIGW) in fly eyes by a GMR-GAL4 driver (GMR > CDGI/LRRK2). For each genotype, images were taken from at least 10 flies. Scale bar, 100 μm. (B) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by SEM. Scale bar, 100 μm. (C) The fly eye size was quantified by ImageJ for each genotype. n = 10. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Light Microscopy, Knockdown, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 5. CDGI acts upstream of LRRK2 to regulate striatal and DA neurodegeneration. (A) Representative images of NeuN staining of mouse striatum. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, the striatal neurons were immuno- labeled by a NeuN antibody. Scale bar, 250 μm. (B) Quantification of NeuN-positive neurons in the striatum using an unbiased stereological method with stereo investiga- tor software from five animals per group. (C) Representative images of DA neuron staining in mouse SNpc. After 9 to 10 months of viral injection, the DA neurons were immunolabeled by a TH antibody. Scale bar, 500 μm. (D) Quantification of TH-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from five to seven animals per group. Data are mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, ** P < 0.01, and ***P < 0.001.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Staining, Injection, Labeling, Software, Immunolabeling

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 6. CDGI acts upstream of LRRK2 to regulate locomotor behavioral deficits. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, a battery of behavioral tests was performed. (A) Open-field test. The total time spent at the center was analyzed. (B) Open-field test. The total time spent at the corner was analyzed. (C) Rotarod test. The average retention time was analyzed. (D) Pole test to monitor behav- ioral abnormalities. The total time to descend to the bottom was recorded. Data are mean ± SEM, n = 5 to 9 per group, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Injection, Battery

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 7. Model of CDGI regulation of LRRK2 function and LRRK2-induced neurodegeneration. LRRK2 GTPase cycle is between inactive off GDP-bound state and active on GTP-bound state. CDGI binds to LRRK2 serving as a physiological GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release, and membrane association and in turn regulates LRRK2-induced striatal and DA neurodegeneration and behavioral deficits.

Article Snippet: LRRK2 GSKI mice were purchased from Taconic (13940) (90).

Techniques: Binding Assay, Activity Assay, Membrane

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: GCNT3 overexpression reduces 5FU resistance in CRC cells. (Panel A) Protein expression levels of GCNT3 in non-infected colorectal cancer (CRC) cells, stable cell lines overexpressing GCNT3 and a battery of shGCNT3. Proteins were detected by western blot using specific antibodies against GCNT3, β-Actin and β-Tubulin, as a loading control. Full-length blots/gels are presented in Supplementary Fig. . (Panel B) mRNA expression levels of GCNT3 measured by RT-QPCR, in non-infected CRC cells, stable cell lines overexpressing GCNT3 and shGCNT3 number 7. Data represent mean ± SEM of three independent experiments. (Panel C) Representative immunofluorescence images of GCNT3 (green) and Tubulin (red) of NoORF, GCNT3, Scrambl and shGCNT3 7 cells. Nuclei were stained with DAPI (blue). Scale bars 50 µm. (Panel D) Comparison of 5-fluoracil (5FU) IC50 values (concentration needed for 50% of viability inhibition) between non-infected CRC cells. Cell viability assays were performed after 72 h treatment. Data represent mean ± SEM of at least two independent experiments each performed in triplicate. (Panel E) Induction of GCNT3 expression by 5FU in CRC cells. Tumour cells were treated with 30 µM 5FU, during 72 h, and their mRNA GCNT3 expression was measured by RT-QPCR and represented in comparison to controls (vehicle-treated cells). Results are expressed as the mean ± SEM. of three independent experiments, each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05). (Panel F) Comparison of 5FU IC50 values between NoORF and GCNT3 cells. Cell viability assays were performed after 72 h treatment. Data represent mean ± SEM of at least three independent experiments each performed in triplicate. Asterisk indicates statistically different values in GCNT3 cells respect to the control (NoORF cells), (*p < 0.05).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Expressing, Infection, Stable Transfection, Battery, Western Blot, Control, Quantitative RT-PCR, Immunofluorescence, Staining, Comparison, Concentration Assay, Inhibition

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: GCNT3 overexpression reduces proliferation, invasion and changes metabolic capacities of CRC cells. (Panel A) xCELLigence proliferation assay of NoORF, GCNT3, Scramble and shGCNT3 7 cells. The rate of proliferation is determined by analyzing the slope of the proliferation line between the 12 and 60 h interval. Complete growth curve of NoORF and GCNT3 SW620 cells from 0 to 72 h after seeding is represented. Results were expressed as 12 h normalized cell index value. Data are represented as mean ± SEM of four independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05). (Panel B) Boyden chamber transwell assay of GCNT3 SW620 cell invasion through Matrigel. After 96 h, SW620 cells were fixed and stained with crystal violet (bottom panels) and counted under an optical microscope. Pictures were taken using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus). Scale bars 100 µm. Data are represented as mean ±SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (**p < 0.01). (Panel C) Oxygen consumption rate (OCR) of NoORF and GCNT3 SW620 cells. Bioenergetics parameters were obtained by adding 2 µM Oligomycin to block ATP-linked OCR, 0.2 µM FCCP to uncouple mitochondria for maximal OCR and 0.5 µM Rotenone/Antimycin A (Rot/AA) to shut down mitochondrial respiration. Right panel reflects the quantification of basal respiration (oxygen consumption used to meet cellular ATP demand, calculated by subtracting non-mitochondrial OCR obtained upon Rot/AA addition) and spare respiratory capacity (capability to respond to an energetic demand, calculated as the difference between maximal and basal OCR). Left panel represents OCR measurements over time for cells stably expressing NoORF or GCNT3. We show representative experiments of 6 measures (n = 3).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Proliferation Assay, Boyden Chamber Transwell Assay, Staining, Microscopy, Software, Blocking Assay, Stable Transfection, Expressing

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Genomic analysis of GCNT3 overexpression. (Panel A) Description of candidate genes selected for validation. (Panel B) Experimental validation of GCNT3 microarray results by qRT-PCR. Bar graph showing the correlation of microarray data with qRT-PCR transcript levels in three CRC cellular models. The X axis shows the selected panel of validated genes and the Y-axis represent the relative fold change by microarray or qRT-PCR. Data represent mean ± SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Biomarker Discovery, Microarray, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Proteomic analysis of GCNT3 overexpression. (Panel A) Immunoprecipitation of NoORF and GCNT3 SW620 cell extracts using V5 antibody. V5-GCNT3 tag protein was detected by Western blot (Inp: Input, IP: Immunoprecipitate, FT; Flow through). Experiments performed in duplicate. Full-length blots/gels are presented in Supplementary Fig. . (Panel B) Protein-protein interaction network of GCNT3 using STRING. Statistically significant interactors of proteomic study were included in the analysis (p-value > 0.0005). Only top-10 statistically significant proteins included in nodes are named, as well as, PAM and CANX.

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Biological processes enriched by GCNT3 overexpression in CRC cells. REViGO Scatterplot of GO categories enriched in GCNT3 genomic (Panel A) and proteomic (Panel B) analysis. GO enrichment analysis for statistically significant transcripts and proteins. The remaining terms after the redundancy reduction were plotted in a two dimensional space. Bubble sizes indicates the p-value (log10 p-value). Semantic space is based on the semantic similarity, which is the degree of relatedness between two entities by measuring the similarity of their annotation meanings. The list of enriched GO terms is subjected to redundancy reduction, based on the “most informative common ancestor” approach in REVIGO and is represented by cluster representatives in a scatterplot. The x- and y-axes of the scatterplot represent the distance between the cluster representatives.

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Over Expression

Journal: Scientific Reports

Article Title: The role of glycosyltransferase enzyme GCNT3 in colon and ovarian cancer prognosis and chemoresistance

doi: 10.1038/s41598-018-26468-4

Figure Lengend Snippet: Clinical relevance of GCNT3 expression in epithelial ovarian cancer (EOC). (Panel A) Association between GCNT3 expression and time to treatment failure (TTF) in EOC. Kaplan–Meier plots for GCNT3 expression in 56 EOC patients. (Panel B) Association of GCNT3 gene expression profiles with response. The expression levels of GCNT3 in non-responders and responders groups are shown. The median value of GCNT3 expression is indicated by the horizontal bar on the graph (Man-Whitney U-test for P values). (Panel C) ROC analysis of the GCNT3 signature in EOC patients. The AUC was 0.667. (Panel D) Forest plot showing the meta-analysis of hazard ratio (HR) and 95% confidence interval (CI) estimates for TTF for the prognostic significance of GCNT3 expression in EOC patients from six different studies. (Panel E) Protein and mRNA expression levels of GCNT3 in non-infected EOC cells and Caov3 stable cell lines overexpressing GCNT3. Proteins were detected by western blot using specific antibodies against GCNT3 and β-Actin. mRNA expression levels of GCNT3 and VEGFA were measured by RT-QPCR. Data represent mean ± SEM of three independent experiments. Student’s t test was applied to assess statistically significant differences (***p < 0.001). Full-length blots/gels are presented in Supplementary Fig. . (Panel F) Boyden chamber transwell assay of GCNT3 Caov3 invasion through Matrigel. After 96 h, Caov3 cells were fixed and stained with crystal violet (bottom panels) and counted under an optical microscope. Pictures were taken using an Olympus CKX41 microscope (Olympus, Tokyo, Japan), with a 20X LCAch objective and registered using analysis getIT software (Olympus). Scale bars 100 µm. On the left, xCELLigence proliferation assay of NoORF and GCNT3 Caov3 cells. The rate of proliferation is determined by analyzing the slope of the proliferation line between the 12 and 60 h interval. Data are represented as mean ± SEM of three independent experiments each performed in triplicate. Student’s t test was applied to assess statistically significant differences (**p < 0.01).

Article Snippet: In clinical samples, GCNT3 gene expression was analyzed using the specific TaqMan probe (Hs00953355_m1, Life Technologies, Carlsbad CA, USA).

Techniques: Expressing, Gene Expression, Infection, Stable Transfection, Western Blot, Quantitative RT-PCR, Boyden Chamber Transwell Assay, Staining, Microscopy, Software, Proliferation Assay

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 1. CDGI interacts with LRRK2. (A) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (B) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from LRRK2 WT and KO mouse whole brains were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (C) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse brain lysates. Lysates prepared from CDGI WT and KO mouse whole brains were subjected to IP with anti-CDGI followed by anti-LRRK2 and anti-CDGI immunoblotting. (D) Co-IP analysis of the interaction between LRRK2 and CDGI in mouse striatal lysates. Lysates prepared from LRRK2 WT and KO mouse stria- tum were subjected to IP with anti-LRRK2 followed by anti-CDGI and anti-LRRK2 immunoblotting. (E) Co-IP analysis of the interaction between MYC-tagged LRRK2 and flag-tagged F1 (the GEF domain), F2 (the EF-hands domain), F3 (the DAG domain), or full-length (FL) CDGI in cotransfected HEK 293T cells. Co-IP with anti-MYC was fol- lowed by anti-flag or anti-MYC immunoblotting. A schematic representation of CDGI F1, F2, and F3 domains is shown. (F) Co-IP analysis of the interaction between V5- tagged CDGI and flag-tagged LRRK2 fragments in cotransfected HEK 293T cells. Co-IP with anti-V5 was followed by anti-flag or anti-V5 immunoblotting. A schematic representation of the different LRRK2 fragments is shown. (G) Co-IP analysis of the interaction between V5-tagged CDGI and MYC-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-V5 immunoblotting. (H) Co-IP analysis of the interaction between flag-tagged CDGI-F1 domain and Myc-tagged LRRK2-WT and TN mutant in cotransfected HEK 293T cells. Co-IP with anti-MYC was followed by anti-flag immunoblotting. (I) Coimmunostaining of CDGI (green), LRRK2 (red), and DARPP32 (magenta) in mouse striatal sections. aa, amino acid.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 2. CDGI acts as a potential GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release. (A) GTP loading analysis of LRRK2 by an in vivo 32P- orthophosphate labeling in HEK 293T cells overexpressing the indicated plasmids. The migration of GTP and GDP is indicated. The Ca2+ ionophore A23187 and TPA were applied. (B) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (C) GTP loading analysis of LRRK2 R1441C (RC) and G2019S (GS) with CDG-WT or GW by an in vivo 32P-orthophosphate labeling in HEK 293T cells. TN (T1348N) was a negative control. (D) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). (E) The levels of LRRK2 bound to GTP were analyzed by pull-down assays with GTP-agarose from HEK 293T cells. (F) Quantification of LRRK2 bound to GTP-agarose beads. Data were normalized to LRRK2-WT alone in (B), (D), and (F). (G) GTP loading analysis of LRRK2 by an in vivo 32P-orthophosphate labeling in WT stria- tal neurons compared to CDGI KO neurons transduced by LV-CDGI-WT or LV-CDGI-GW. (H) Quantification of LRRK2 GTP binding activity by the ratio GTP to (GTP + GDP). Data were normalized to WT neurons. (I) A diagram of the guanine nucleotide exchange assay is illustrated. Free BODIPY-FL-GDP is quenched with a low fluorescence in the solutions while showing increased fluorescence upon binding to GTPases. GEF catalyzes the exchange of preloaded BODIPY-FL-GDP for GTP causing a decrease in fluorescence. (J) Recombinant LRRK2 preloaded with BODIPY-FL-GDP was incubated with recombinant GST-CDGI proteins in the presence of excess cold GTP. Nucleotide exchange on LRRK2 was monitored by the fluorescence intensity change with different concentrations of CDGI-WT every 36 s for 15 min. Data are the means ± SEM, n = 3. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not statistically significant.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Binding Assay, Activity Assay, In Vivo, Labeling, Migration, Negative Control, Fluorescence, Recombinant, Incubation

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 3. CDGI increases LRRK2 membrane association. (A to C) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in HEK 293T cells co- transfected with LRRK2 and CDGI-WT or GW. After 48 hours, cells were harvested and fractionated into cytosol and membrane fractions. Samples were immunoblotted with anti-MYC, V5, LAMP1, Rab10, and phosphor-Rab10. Data were normalized to LRRK2-MYC alone. (D and E) Liquid nitrogen freeze-thaw methods assessing LRRK2 membrane association. HEK 293T cells with stably expressed eGFP-LRRK2 were transfected with or without mCherry-CDGI. After 48 hours, cells were permeabilized by liquid nitrogen freeze-thaw to deplete cytosol and then fixed, immunostained with LAMP1, and visualized by confocal microscopy. Scale bar, 20 μM. Data were normalized to eGFP-LRRK2 stable cells transfected with mCherry. (F and G) Cellular fractionation assay of LRRK2 intensity on membrane and cytosol fractions in CDGI WT striatal neu- rons compared to CDGI KO neurons. CDGI WT and KO striatal neurons were treated with A23187 and TPA before being harvested and fractionated into cytosol and mem- brane fractions. Data were normalized to CDGI-WT neurons. Data are the means ± SEM, n = 3. One-way ANOVA followed by Tukey’s post hoc test was used for the data analysis of multiple comparisons, and Student’s t tests (unpaired, two-tailed) were used for the data analysis of two comparisons. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Membrane, Cell Fractionation, Transfection, Stable Transfection, Confocal Microscopy, Two Tailed Test

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 4. CDGI acts upstream of LRRK2 to regulate retinal neurodegeneration. (A) Representative images of eye morphology of 1-week-old flies of the indicated geno- types by light microscopy. Drosophila LRRK2 (dLRRK) RNAi knockdown (dLRRKRI), human LRRK2 WT (LRRK2WT), and LRRK2 mutant GS or RC (LRRK2GS or LRRK2RC) were coex- pressed with human CDGI WT (CDGIWT) or CDGI-GW (CDGIGW) in fly eyes by a GMR-GAL4 driver (GMR > CDGI/LRRK2). For each genotype, images were taken from at least 10 flies. Scale bar, 100 μm. (B) Representative images of eye morphology of 1-week-old flies of the indicated genotypes by SEM. Scale bar, 100 μm. (C) The fly eye size was quantified by ImageJ for each genotype. n = 10. Data are mean ± SEM, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, **P < 0.01, and ****P < 0.0001.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Light Microscopy, Knockdown, Mutagenesis

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 5. CDGI acts upstream of LRRK2 to regulate striatal and DA neurodegeneration. (A) Representative images of NeuN staining of mouse striatum. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, the striatal neurons were immuno- labeled by a NeuN antibody. Scale bar, 250 μm. (B) Quantification of NeuN-positive neurons in the striatum using an unbiased stereological method with stereo investiga- tor software from five animals per group. (C) Representative images of DA neuron staining in mouse SNpc. After 9 to 10 months of viral injection, the DA neurons were immunolabeled by a TH antibody. Scale bar, 500 μm. (D) Quantification of TH-positive neurons in SNpc using an unbiased stereological method with stereo investigator software from five to seven animals per group. Data are mean ± SEM, two-way ANOVA followed by Tukey’s post hoc test, *P < 0.05, ** P < 0.01, and ***P < 0.001.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Staining, Injection, Labeling, Software, Immunolabeling

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 6. CDGI acts upstream of LRRK2 to regulate locomotor behavioral deficits. Ten-month-old WT, LRRK2 GSKI, RCKI, and KO mice were injected with AAV1-CDGI-WT or GW into the striatum. After 9 to 10 months of viral injection, a battery of behavioral tests was performed. (A) Open-field test. The total time spent at the center was analyzed. (B) Open-field test. The total time spent at the corner was analyzed. (C) Rotarod test. The average retention time was analyzed. (D) Pole test to monitor behav- ioral abnormalities. The total time to descend to the bottom was recorded. Data are mean ± SEM, n = 5 to 9 per group, one-way ANOVA followed by Tukey’s post hoc test, *P < 0.05.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Injection, Battery

Journal: Science advances

Article Title: CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

doi: 10.1126/sciadv.adn5417

Figure Lengend Snippet: Fig. 7. Model of CDGI regulation of LRRK2 function and LRRK2-induced neurodegeneration. LRRK2 GTPase cycle is between inactive off GDP-bound state and active on GTP-bound state. CDGI binds to LRRK2 serving as a physiological GEF for LRRK2 to increase LRRK2 GTP binding activity and GDP release, and membrane association and in turn regulates LRRK2-induced striatal and DA neurodegeneration and behavioral deficits.

Article Snippet: LRRK2- WT- MYC was from the Dawson lab (Addgene plasmid #17609) (11).

Techniques: Binding Assay, Activity Assay, Membrane