arf6 pulldown activation assay kit  (Cytoskeleton Inc)

Journal: iScience

Article Title: TBC1D24 interacts with the v-ATPase and regulates intraorganellar pH in neurons

doi: 10.1016/j.isci.2024.111515

Figure Lengend Snippet:

Article Snippet: Arf6 Activation Assay Biochem Kit , Cytoskeleton , Cat#BK033-S.

Techniques: Virus, Expressing, Recombinant, Protease Inhibitor, Activation Assay, Software

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A Rab10–ACAP1–Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling

doi: 10.1007/s00018-023-04722-x

Figure Lengend Snippet: Endocytosis and recycling profiles of M4 mutants

Article Snippet: Arf6 activity was measured using an Arf6 Pull-Down Activation Assay Biochem kit (BK033-S; Cytoskeleton), according to the manufacturer’s instruction.

Techniques:

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A Rab10–ACAP1–Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling

doi: 10.1007/s00018-023-04722-x

Figure Lengend Snippet: CCh-activated and internalized EGFP-M4 is mainly hindered in the Rab11-REs in Arf6 T27N-expressing cells. a Representative confocal micrographs showing the subcellular localization of EGFP-M4 before or after CCh stimulation and after removal of CCh in cells expressing wild type, Q67L, or T27N Arf6-RFP. Transfected cells were untreated (left) or treated (middle) with CCh for 15 min or pretreated with CCh for 60 min followed by 60 min washout (right). Insets show the magnified boxed regions. Scale bar, 10 μm. b–d Quantification of the surface expression level (b), internalization (c) and recycling efficiency (d) of M4 receptor as measured by images shown in (a). Data are displayed as mean ± SEM, n ≥ 30 cells from three independent experiments, Kruskal–Wallis test with Dunn’s post hoc test in (b). Data represent mean ± SD from three independent experiments, one-way ANOVA followed by Dunn’s post hoc test in (c and d). ns not significant; ****p < 0.0001. e Quantification of the recycling efficiency of HA-tagged M4 receptors using ELISA. HEK293 cells co-expressing Arf6 variants or empty vector were treated with CCh and followed by washout procedure. Data are presented as the percentage of the cell surface M4 expression level compared with vehicle group and shown as mean ± SD from 3 independent experiments. One-way ANOVA followed by Dunn’s post hoc test, ns not significant; ***p < 0.001. f Manders’ coefficients for fraction of EGFP-M4 overlapping Arf6-RFP variants as depicted in (a) were calculated. Data are displayed as mean ± SEM, n ≥ 20 cells from three independent experiments, Kruskal–Wallis test with Dunn’s post hoc test. ns not significant; ****p < 0.0001. g Representative confocal micrographs showing the overlap of internalized M4 with mCh-Rab5 (left) or mCh-Rab11 (right) after removal of CCh in Arf6 T27N co-expressing cells. Fluorescence intensity profiles are shown for the indicated region and path. Scale bar, 5 μm. h Colocalization of M4 receptors with Rab5 or Rab11 in Arf6 T27N-expressing cells as depicted in (g) was calculated. Data are displayed as mean ± SEM. n ≥ 30 cells from three independent experiments. Unpaired two-tailed Student’s t test. ****p < 0.0001. Nuclear boundaries are demarcated with dashed lines. Cell boundaries in (g) are demarcated with solid lines

Article Snippet: Arf6 activity was measured using an Arf6 Pull-Down Activation Assay Biochem kit (BK033-S; Cytoskeleton), according to the manufacturer’s instruction.

Techniques: Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Fluorescence, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A Rab10–ACAP1–Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling

doi: 10.1007/s00018-023-04722-x

Figure Lengend Snippet: ACAP1 is the Arf6 GAP and negatively regulates M4 recycling. a Representative confocal images showing the prominent translocation of Arf6-RFP from the PM to the cytoplasm in cells expressing EGFP-ACAP1, but not in cells expressing EGFP-ACAP2, ACAP1 R448Q or empty vector. Scale bar, 10 μm. b Quantification of the percentage of cells with PM Arf6 or cytoplasmic Arf6 in each condition shown in (a). Data are displayed as mean ± SD. n = 3 independent experiments (≥ 40 cells). Unpaired two-tailed Student’s t test. ns not significant; ***p < 0.001. c Confocal images showing the subcellular distribution of EGFP-ACAP1 relative to Arf6 Q67L-RFP or Arf6 T27N-RFP. Transfected cells were untreated or treated with 50 μg/ml digitonin for 5 min at RT before image taken. Scale bar, 10 μm. d Quantification of the Pearson’s correlation coefficients for ACAP1 with Arf6 Q67L or Arf6 T27N after digitonin treatment as depicted in (c). Data are displayed as mean ± SEM, n ≥ 30 cells from three independent experiments. Unpaired two-tailed Student’s t-test. ****p < 0.0001. e and f ACAP1 promotes Arf6 inactivation in HEK293 cells. e Cells were co-transfected with Arf6-Flag and EGFP-tagged ACAP1, ACAP1 R448Q, or empty vector. The transfected cells were lysed to analyze the protein level of activated Arf6 (Arf6-GTP) using the GST-GGA3 PBD assay. The cells only transfected with Arf6-Flag and lysed in the presence of 200 μM GTPγS or 1 mM GDP was used as positive or negative control, respectively. Elutes were analyzed by SDS-PAGE followed by immunoblotting with anti-Arf6 antibody. f Three independent experiments of the pulled-down Arf6-GTP level shown in (e) were analyzed for the gray value of protein bands. Data are displayed as mean ± SD. One-way ANOVA followed by Dunnett’s post hoc test. ****p < 0.0001. g Confocal micrographs showing the recycling of EGFP-M4 after removal of CCh is impaired in cells expressing mCh-ACAP1. Scale bar, 10 μm. h Quantification of the recycling efficiency of EGFP-M4 in each group indicated in (g). Data are displayed as mean ± SD. n = 3 independent experiments. Unpaired two-tailed Student’s t test. ***p < 0.001. i Quantification of the recycling efficiency of HA-tagged M4 receptors using ELISA. HEK293 cells co-expressing ACAP1 or empty vector were treated with CCh and followed by washout procedure. Data are presented as the percentage of the cell surface M4 expression level compared with vehicle group and shown as mean ± SD from 3 independent experiments. Unpaired two-tailed Student’s t test. ****p < 0.0001. Nuclear boundaries are demarcated with dashed lines. Cell boundaries in (c) are demarcated with solid lines

Article Snippet: Arf6 activity was measured using an Arf6 Pull-Down Activation Assay Biochem kit (BK033-S; Cytoskeleton), according to the manufacturer’s instruction.

Techniques: Translocation Assay, Expressing, Plasmid Preparation, Two Tailed Test, Transfection, Negative Control, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A Rab10–ACAP1–Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling

doi: 10.1007/s00018-023-04722-x

Figure Lengend Snippet: The GAP activity of ACAP1 is indispensable for Rab10-GTP resultant Arf6 inactivation and defective M4 recycling. a ACAP1-Rab10 interaction was assessed in lysates of HEK293 cells transiently expressing HA-ACAP1 and 3 × Flag-tagged Rab10 WT, Q68L, or T23N mutant, respectively. Lysates were IP through the HA epitope followed by immunoblotting using anti-Flag and anti-HA antibodies. b Confocal images showing the subcellular distribution of ACAP1 in indicated context. Transfected cells were untreated or treated with 50 μg/ml digitonin for 5 min at RT before image taken. Insets show the expression of mCh-Rab10 variants (− Digitonin) or the magnified boxed regions (+ Digitonin). Scale bar, 10 μm. c Quantification of the Pearson’s correlation coefficients for ACAP1 with Rab10 variants after digitonin treatment as depicted in (b). Data are displayed as mean ± SEM, n ≥ 30 cells from three independent experiments, Kruskal–Wallis test with Dunn’s post hoc test. ***p < 0.001; ****p < 0.0001. d and e Quantification of the average Fluorescence intensity (d) and puncta number (e) of ACAP1 in digitonin-treated cells as (b) indicates. Data are displayed as mean ± SEM. n ≥ 30 cells from three independent experiments. Kruskal–Wallis test with Dunn’s post hoc test. ns not significant; ****p < 0.0001. f Expressing Rab10 Q68L reduces the Arf6-GTP amount in HEK293 cells. Cells were co-transfected with Arf6-Flag and HA-Rab10 Q68L or control empty vector. The protein level of activated Arf6 (Arf6-GTP) was assessed using the GST-GGA3 PBD assay. Elutes were analyzed by SDS-PAGE followed by immunoblotting with anti-Arf6 antibody. g Three independent experiments of the pulled-down Arf6-GTP level shown in (f) were analyzed for the gray value of protein bands. Data are displayed as mean ± SD. Unpaired two-tailed Student’s t test. **p < 0.01. h Rab10 Q68L resultant defective M4 recycling is reversed by co-expressing ACAP1 R448Q. HEK293 cells were co-transfected with EGFP-M4, BFP-Rab10 Q68L, and mCherry-tagged ACAP1, ACAP1 R448Q, or control empty vector. Boxed areas are magnified into separate channels on the right. G/R/B represents the green/red/blue channel, respectively. Scale bar, 10 μm. i Quantification of the percentage of cells with PM M4 or cytoplasmic M4 in each condition shown in (h). Data are displayed as mean ± SD. n = 3 independent experiments (≥ 30 cells). Unpaired two-tailed Student’s t-test. ns not significant; ****p < 0.0001. Nuclear boundaries are demarcated with dashed lines. Cell boundaries in (b) are demarcated with solid lines

Article Snippet: Arf6 activity was measured using an Arf6 Pull-Down Activation Assay Biochem kit (BK033-S; Cytoskeleton), according to the manufacturer’s instruction.

Techniques: Activity Assay, Expressing, Mutagenesis, Western Blot, Transfection, Fluorescence, Plasmid Preparation, SDS Page, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A Rab10–ACAP1–Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling

doi: 10.1007/s00018-023-04722-x

Figure Lengend Snippet: Disruption of Rab10/Arf6 cascade affects M4-mediated Ca2+ signal resensitization. a Schematic diagram illustrates Fluo-4-based intracellular calcium assay. b Color-coded fluorescence intensity changes show the dynamics of intracellular Ca2+ in response to CCh application in CHO-K1 cells expressing RFP-M4 (lower) or not (upper). Red indicates a large increase in Ca2+; blue indicates little increase in Ca2+. Scale bar, 10 μm. c Average normalized curves for Fluo-4 fluorescence intensity change upon CCh stimulation in cells expressing RFP-M4 or not. The original or resensitizing Ca2+ signal was induced by CCh administration as denoted in (a). d Effect of PTX treatment on the CCh-induced Fluo-4 fluorescence intensity change in M4-expressing cells. Co-cultured RFP-M4-expressing and plain cells were left untreated (- PTX) or pre-treated with 100 ng/ml PTX for 12 h (+ PTX) and administrated with CCh to activate M4. e and f Analysis of the fold increase of peak intracellular Ca2+ as depicted in (c) and (d), respectively. Data represent the mean ± SD of at least five independent experiments. One-way ANOVA followed by Tukey’s post hoc test (c). Two-way ANOVA followed by Tukey’s post hoc test (d). ns not significant; ***p < 0.001; ****p < 0.0001. g The effect of Rab10 Q68L on the resensitizing Ca2+ signal in M4 WT and ΔR386-A393 mutant-expressing cells. Resensitizing Ca2+ flux was initiated by repeated CCh treatment and examined as illustrated in diagram (a). h Histograms showing the fold increase of peak intracellular Ca2+ in each indicated condition. Data represent the mean ± SD of at least five independent experiments. One-way ANOVA followed by Tukey’s post hoc test. ns not significant; **p < 0.01

Article Snippet: Arf6 activity was measured using an Arf6 Pull-Down Activation Assay Biochem kit (BK033-S; Cytoskeleton), according to the manufacturer’s instruction.

Techniques: Disruption, Calcium Assay, Fluorescence, Expressing, Cell Culture, Mutagenesis

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: A Rab10–ACAP1–Arf6 GTPases cascade modulates M4 muscarinic acetylcholine receptor trafficking and signaling

doi: 10.1007/s00018-023-04722-x

Figure Lengend Snippet: A model of the GTPase cascade between Rab10 and Arf6 via ACAP1 controlling M4 mAChR transit through endosomes. Rab10 is recruited to the Arf6-positive sorting endosomes and recruits its effector, Arf6 GAP-ACAP1, to the same compartment. ACAP1 then inactivates Arf6. Agonist-activated and internalized M4 receptor binds to the GTP-Rab10, hence over activation of Rab10 leads to hampered recycling of M4 on Rab5-positive endosomes. Aberrant inactivation of Arf6, however, hinders the recycling of M4 predominantly on Rab11-positive endosomes. M4 receptor missing the R386-A393 motif will bypass the control of Rab10 and directly returns to the PM via Rab4-positive early endosomes

Article Snippet: Arf6 activity was measured using an Arf6 Pull-Down Activation Assay Biochem kit (BK033-S; Cytoskeleton), according to the manufacturer’s instruction.

Techniques: Activation Assay

Journal: PLoS ONE

Article Title: Differential Regulation of Rab GTPase Expression in Monocyte-Derived Dendritic Cells upon Lipopolysaccharide Activation: A Correlation to Maturation-Dependent Functional Properties

doi: 10.1371/journal.pone.0073538

Figure Lengend Snippet: Quantitative RT-PCR shows expression levels of Rab5a (A), Rab8a (B), Rab35 (C), Arf6 (D) and Rab21 (E) in moDCs, after LPS stimulation for the indicated time periods. All data are normalized to initial iDC expression levels. Means and SD are shown, n = 6, * P<0.05.

Article Snippet: To assess Arf6 activation, cell lysis, pull-down and detection was performed according to the protocol and with reagents supplied by the manufacturer (Cat. # BK033-S, Cytoskeleton, Inc., Denver, CO, USA).

Techniques: Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Differential Regulation of Rab GTPase Expression in Monocyte-Derived Dendritic Cells upon Lipopolysaccharide Activation: A Correlation to Maturation-Dependent Functional Properties

doi: 10.1371/journal.pone.0073538

Figure Lengend Snippet: (A) Protein lysate from moDCs stimulated with LPS for the indicated time points were run on SDS-PAGE and subjected to Western blot analysis using anti-Rab7b, anti-Rab27a, anti-Rab21, anti-Arf6, anti-Rab9 and anti-tubulin antibodies. (B) The intensity of the bands was quantified by densitometry, and normalized against tubulin. Protein levels are relative to the initial iDC levels. Mean and SD are shown, n= 3.

Article Snippet: To assess Arf6 activation, cell lysis, pull-down and detection was performed according to the protocol and with reagents supplied by the manufacturer (Cat. # BK033-S, Cytoskeleton, Inc., Denver, CO, USA).

Techniques: SDS Page, Western Blot