Journal: Journal of Biological Chemistry

Article Title: A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

doi: 10.1074/jbc.m404196200

Figure Lengend Snippet: FIG. 1. Binding of AMAP2 with GTP-Arf6. A and B, 5 g of GST-ArfGAP (A), GST-GGA3 (B), or GST alone, each produced in E. coli and purified on glutathione-beads, were incubated with 250 g of COS-7 cell lysates expressing various HA-Arfs, as indicated, and proteins co-precipitated with the GST fusion proteins were analyzed by immunoblotting. C, COS-7 cells were transfected with pcDNA-Arf6-HA and radiolabeled with [32P]orthophosphate. Cell lysates were then incubated with 5 g each of GST-ArfGAP (lane 1) or GST alone (lane 2), and bound radioactivities were analyzed by thin layer chromatography, as described under “Experimental Procedures.” As a control, HA-Arf6 was immuno- precipitated using an anti-HA antibody and similarly analyzed (lane 3). A representative result is shown from four independent experiments. D, 0.3 g of pcDNA-Arf-HA was co-transfected with 4 g of pEBG-AMAP2 or with 2 g of pEBG vector (for GST alone) plus 2 g of pcDNA empty vector in COS-7 cells, as indicated. GST proteins were then pulled down from 500 g of the cell lysates, prepared in 1% Nonidet P-40 buffer in the absence or presence of 10 mM Mg2, and analyzed for the co-precipitation of Arf isoforms. In A, B, and D, tagged proteins were detected by antibodies against tags, as indicated. The total includes 20 g of total cell lysate.

Article Snippet: Mouse monoclonal anti-Arf6 antibody and rabbit polyclonal anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Binding Assay, Produced, Purification, Incubation, Expressing, Western Blot, Transfection, Thin Layer Chromatography, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

doi: 10.1074/jbc.m404196200

Figure Lengend Snippet: FIG. 3. Subcellular localization of AMAP2 in HeLa cells. A, cells were fixed in methanol for 5 min at 20 °C and labeled with rabbit AMAP2 polyclonal antibodies coupled with a Cy2-conjugated anti-rabbit IgG antibody (a), or live cells expressing EGFP-AMAP2 at a low level were analyzed by detecting the autofluorescence from the tag (b). B, 1 g each of pcDNA-Xpress-amphiphysin IIm and pEGFP-AMAP2 were co-transfected with 0.5 g of pcDNA-Arf6-HA and its mutants (a–p) or pcDNA-Arf1-HA and its mutants (q–x), as indicated. HA-Arfs and Xpress-amphiphysin IIm were detected using an HA polyclonal antibody coupled with a Cy5-conjugated anti-rabbit IgG antibody and an anti-Xpress monoclonal antibody coupled with a Cy3-conjugated anti-mouse IgG antibody, respectively. EGFP-AMAP2 was detected by its autofluorescence. The right panels show merged views of the left three images. C, protein colocalization was quantified as indicated, according to a method described under “Experimental Procedures.” Results were shown as mean values S.E. from three independent experiments. D, cells transfected with 4 g of pEGFP-AMAP2 and 2 g of pcDNA-Arf6Q67L-HA were subjected to immunoelectron microscopy analysis using with anti-HA (a) or anti-GFP antibodies (b). E, cells were transfected with 0.5 g of pcDNA-Arf6Q67L-HA (a–d) or pcDNA-wild type Arf6-HA (e–h), as indicated. Twenty-four h later, cells were fixed with paraformaldehyde. Endogenous AMAP2 and amphiphysin IIm were detected by an anti-AMAP2 rabbit polyclonal antibody coupled with a Cy2-conjugated anti-rabbit IgG antibody or an anti-amphiphysin IIm rat monoclonal antibody coupled with a Cy3-conjugated anti-rat IgM antibody. Arf6-HA proteins were detected using an HA monoclonal antibody coupled with a Cy5-conjugated anti-mouse IgG antibody. The right panels show merged views of the left three images. F, cells were transfected with 0.5 g of pcDNA-Arf6-HA. Twenty-four h after transfection, cells were starved of serum for another 24 h (a–c) and then stimulated with 10 ng/ml epidermal growth factor for 7 min (d–f) before fixation with paraformaldehyde. Endogenous AMAP2 was detected by an AMAP2 rabbit polyclonal antibody coupled with a Cy2-conjugated anti-rabbit IgG antibody. Arf6-HA proteins were detected using an HA monoclonal antibody coupled with a Cy5-conjugated anti-mouse IgG antibody. The right panels show merged views of the left two images. Antibodies that can clearly label endogenous Arf6 in HeLa cells were not available. In B, D, E, and F, images were acquired using laser confocal microscopy by adjusting the focus 3.0 m above the surface of the glass chamber plate, which was near the center of the nucleus of the majority of cells. Bars, 10 m in A, B, E, and F; 200 nm in D.

Article Snippet: Mouse monoclonal anti-Arf6 antibody and rabbit polyclonal anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Labeling, Expressing, Transfection, Immuno-Electron Microscopy, Confocal Microscopy

Journal: Journal of Biological Chemistry

Article Title: A Novel Mode of Action of an ArfGAP, AMAP2/PAG3/Papα, in Arf6 Function

doi: 10.1074/jbc.m404196200

Figure Lengend Snippet: FIG. 4. Involvement of AMAP2 in Tac internalization in HeLa cells. A–C, cells were treated with 50 nM of siRNA oligonucleotide duplexes, each designed for Arf6, AMAP2, or APAP2 silencing, as indicated. Twelve h later, the cells were transfected with 0.4 g of the pKCR-Tac plasmid and 0.1 g of pEGFP-C1 and incubated for a further 24 h before being subjected to endocytosis assays using an anti-Tac antibody (B) or biotinylated Tfn (C) as probes. Oligonucleotides with an irrelevant sequence (irr) were included as a negative control. Protein knockdown was assessed by immunoblotting, as indicated (A). siRNA treatment did not affect protein expression levels of Tac (data not shown). D and E, cells were co-transfected with 0.4 g of pKCR-Tac and 1.6 g of plasmids encoding EGFP-AMAP2 (AMAP2), its ArfGAP do- main deletion mutant (ArfGAP), EGFP-APAP2 (APAP2), K44A mu- tant of EGFP-dynamin-2 (Dyn (K44A)), or EGFP alone, as indicated. They were then subjected to endocytosis assays of Tac (D) and Tfn (E). For co-overexpression of wild type Arf6-HA together with EGFP- AMAP2, 0.3 g of pcDNA-Arf6-HA was included (AMAP2 Arf6). Endocytic blockage in transfection-positive cells was defined as de- scribed under “Experimental Procedures.” Results are means S.E. from three independent experiments. *, p 0.01 against values of the control.

Article Snippet: Mouse monoclonal anti-Arf6 antibody and rabbit polyclonal anti-HA antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Incubation, Sequencing, Negative Control, Knockdown, Western Blot, Expressing, Mutagenesis, Over Expression, Control

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Membrane ABCA1 level decreased in ApoE4 primary astrocytes compared with ApoE3. A, ABCA1 mRNA level in ApoE3 and ApoE4 immortalized astrocytes was assessed by qRT-PCR. B, Total ABCA1 protein in ApoE3 and ApoE4 immortalized astrocytes were detected by Western blot. C, Densitometric quantification total ABCA1 protein of blotting shown in B from three independent experiments. D, ABCA1 mRNA level in ApoE3 and ApoE4 primary astrocytes was assessed by qRT-PCR. E, Membrane (MEM) and total ABCA1 and ApoE protein in ApoE3 and ApoE4 primary astrocytes were detected by Western blot. Na/K+ ATPase was used as the membrane protein loading control. In this immunoblot, the membrane fraction loaded was prepared from 3/8 of total cell volume. The loaded total fraction was 1/10 of total cell lysate volume. Actin was used as the total protein loading control. F, G, Densitometric quantification of membrane (F) and total (G) ABCA1 and ApoE protein of blotting shown in (E) from three independent experiments. H, ARF6 protein in ApoE3 and ApoE4 primary astrocytes was detected by Western blot. I, Densitometric quantification of blotting shown in (H) from three independent experiments. ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Quantitative RT-PCR, Western Blot

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Effect of ApoE isoforms on ABCA1-mediated cholesterol efflux and ApoE lipidation. A, B, ApoE3 or ApoE4 primary astrocyte were seeded overnight in 96-well plates and then incubated with 3H-labeled cholesterol for 24 h. After washing with PBS, DMEM containing 2 mg/ml BSA were added to the wells and cholesterol efflux was measured at 4 h (A) and 24 h (B). Values showed are normalized to the ApoE3 group. C, ApoE4 mouse primary astrocytes were treated with ARF6 siRNA for 48 h and ARF6 expression was measured by Western blot. D, E, ApoE4 primary astrocyte were treated with ARF6 siRNA followed by cholesterol efflux measurement at 4 h (D) and 24 h (E). (F, G) BHK cells were labeled with 3H-cholesterol followed by induction of ABCA1 expression, and then cholesterol efflux was determined after treatment with 0.2 μm recombinant ApoE3 or ApoE4 (F) or with 15 μl of CSF from non-ε4 (n = 9), ε3/ε4 (n = 8), and ε4/ε4 carriers (n = 3) (G) for 4 h. H, ApoE HDL particles in astrocyte conditioned medium (ACM) from ApoE3 or ApoE4 immortalized astrocytes were analyzed by native-PAGE followed by anti ApoE Western blot. ApoE particles were separated into five distinct sizes, very large (α0>1000KDa), large (α1∼720 KDa), medium (α2∼480 KDa), small (α3∼242 KDa) and very small (α4∼35 KDa). I–M, Densitometric quantification of different size of ApoE particles of blotting shown in H from three independent experiments. **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Incubation, Labeling, Expressing, Western Blot, Recombinant, Clear Native PAGE

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Effect of CS-6253 on ABCA1 expression and trafficking. A, ApoE4 primary astrocytes were treated with CS-6253 peptide (1 μm) for 4 h and then the membrane and total cellular protein were collected. ABCA1, ApoE, and ARF6 were detected by Western blot. B, C, Quantification of membrane ABCA1 and ApoE (B) and total ABCA1 and ARF6 (C) by Western blot shown in A from three independent experiments. D, ApoE4 immortalized astrocytes were treated with CHX with and without CS-6253 for 2 and 4 h. ABCA1 protein expression in cell lysates was detected by Western blot. E, Quantification of Western blot shown in D from three independent experiments. ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Expressing, Western Blot

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Effect of CS-6253 on cholesterol efflux function. A, ApoE4 primary astrocytes were incubated with 3H-labeled cholesterol for 24 h. The cells were induced to express ABCA1 by GW3965 compound for 18 h. After washing with PBS, cholesterol efflux were determined after treatment with or without CS-6253 (1 μm) for 4 h. B, ApoE4 mouse primary astrocytes were treated with ARF6 siRNA or scramble siRNA followed by labeling with 3H-cholesterol for 24 h. After washing with PBS, cholesterol efflux was measured after the addition of CS-6253 for 4 h. C, D, BHK cells were labeled with 3H-cholesterol and ABCA1 expression was induced by mifepristone for 18 h. Cholesterol efflux was measured after treatment with 0.2 μm rApoE4 with or without 1 μm CS-6253 (C) or after treatment with 1 μm CS-6253 plus CSF from ε3/ε3 (n = 7), ε3/ε4 (n = 8), or ε4/ε4 (n = 3) carriers (D) for 4 h. **p < 0.01; ***p < 0.001.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques: Incubation, Labeling, Expressing

Journal: The Journal of Neuroscience

Article Title: ApoE4 Alters ABCA1 Membrane Trafficking in Astrocytes

doi: 10.1523/JNEUROSCI.1400-19.2019

Figure Lengend Snippet: Illustration showing the interaction of ApoE isoforms with ABCA1. A, The interaction of ApoE with ABCA1 favors lipidation of ApoE to the form ApoE HDL. B, ApoE4 is prone to forming protein aggregates and activates ARF6 that in turn decreases ABCA1 recycling; lowering ABCA1-mediated lipidation of ApoE. CS-6253 promotes ABCA1 recycling to the plasma membrane and favors ApoE lipidation, counteracting the effects of ApoE4.

Article Snippet: The following antibodies were used: anti-human ApoE antibody (Academy Bio-Medical, catalog #A14 50A-G1b), anti-ABCA1 antibody (Abcam, catalog #ab18180), anti-β-actin antibody (CST, catalog #3700), anti-ARF6 antibody (CST, catalog #5740), anti-β-Amyloid (6E10, BioLegend, catalog #803001), HRP-linked anti-mouse IgG (CST, catalog #7076), HRP-linked anti-rabbit IgG (CST, catalog #7074), HRP-linked anti-goat IgG (Santa Cruz Biotechnology, catalog #sc-2020).

Techniques:

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: Cytohesins are required to promote HGF-induced activation of Rac1 and Arf6 in injured kidney. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and stained either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Staining

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: HGF-induced activation of Rac1 and Arf6 in injured kidney occurs mainly at the proximal tubules. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both every 24 h for 96 h. Injured kidneys were then harvested, cryosectioned (10 mm), and incubated with either against active Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with rabbit anti aquaporin-1. Next, sections were stained with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 546. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Incubation, Staining

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: HGF induces the activation of Rac1 and Arf6 in damaged kidneys early after reperfusion. Mice were subjected to IRI and treated with either HGF, SecinH3 or a combination of both 24 h after reperfusion. Injured kidneys were then harvested 24 h after treatment, cryosectioned (10 mm), and stained either against GTP-Arf6 (panels A–D) or GTP-Rac1 (panels E–H), and costained with Alexa Fluor 647 phalloidin to detect F-actin. Tissues derive from: (A and E) IRI-untreated mice, (B and F) SecinH3-treated IRI mice, (C and G) HGF-treated IRI mice or, (D and H) HGF and SecinH3-treated IRI mice. The scale bars represent 100 μ m.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Staining

Journal: Physiological Reports

Article Title: The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

doi: 10.14814/phy2.12442

Figure Lengend Snippet: Cytohesin-dependent Arf6 to Rac1 signaling module is required to promote the HGF-stimulated recovery of damaged kidney. Treatment of IRI kidneys with HGF activates the HGF receptor c-met, resulting in a signaling cascade event that leads to activation of the Arf GEFs cytohesins. Active cytohesins promote Arf6 to exchange its bound GDP molecule for GTP, resulting in its activation. GTP-bound Arf6 thus activates Rac1. Activation of Rac1 induces epithelial cell migration to the bare areas of the tubules, promoting damaged kidney recovery.

Article Snippet: Samples were then washed three times with PBS for 5 min each and blocked in PBS containing 1% BSA for 1 h. Next, sections were incubated with primary antibodies specific for Arf6-GTP (Anti-Active Arf6 Mouse Monoclonal Antibody, catalog # 26918 NewEast Biosciences, Malvern, PA) or against Rac1-GTP (Anti-Active Rac1-GTP Mouse Monoclonal Antibody catalog # 26903, NewEast Biosciences) plus or minus primary antibody against aquaporin 1 (Aqp1) (Millipore).

Techniques: Activation Assay, Migration

Journal: bioRxiv

Article Title: Endothelial specific reduction in Arf6 impairs insulin-stimulated vasodilation and skeletal muscle blood flow resulting in systemic insulin resistance

doi: 10.1101/2023.05.02.539173

Figure Lengend Snippet: (A, B) Generation and identification of endothelial cell (EC) specific tamoxifen inducible Arf6 knockout(iECKO) mouse model, (C) gene expression of Arf6 relative to 18S in EC-enriched carotid artery trizol effluents, lung, liver, muscle, white adipose tissue (WAT) and kidney 14 days after tamoxifen administration (4mg/day, 4 days, oral gavage), N=5-6/group, (D, E) representative western blotting images and densitometric quantification of Arf6 and β-actin proteins from primary lung endothelial cells (N=4/group. replicates were pooled from 3 mice) collected 14 days after tamoxifen administration. Data are shown as mean ± SEM with individual datapoints. Group difference was assessed using independent Student’s t test. *p≤0.02 vs WT.

Article Snippet: Protein expression was measured by standard western blot procedures using anti-mouse primary antibodies against Arf6 (rabbit monoclonal; 1:1000; 19kDa; Cell Signaling), β-actin (mouse monoclonal; 1:1000; 40kDa; Abcam), total Akt (pan-Akt; 1:1000; 60 kDa; cell signaling), phosphorylated Akt (s-473 p-Akt; 1:1000; 60 kDa; cell signaling), phosphorylated eNOS; 1:1000; cell signaling).

Techniques: Knock-Out, Gene Expression, Western Blot

Journal: bioRxiv

Article Title: Endothelial specific reduction in Arf6 impairs insulin-stimulated vasodilation and skeletal muscle blood flow resulting in systemic insulin resistance

doi: 10.1101/2023.05.02.539173

Figure Lengend Snippet: (A) Body mass of the wildtype (WT) and tamoxifen inducible endothelial Arf6 knockout (iECKO) mice before and after tamoxifen administration. (B) gastrocnemius muscle mass and white adipose tissue mass of WT and iECKO mice, (C) blood glucose response curves during a glucose tolerance test (GTT), (D) Plasma insulin level at baseline (6h fasting) and during GTT, (E) homeostatic model assessment for insulin resistance (HOMA-IR), (F) homeostatic model assessments for beta cell function (HOMA-B%), (G, H) blood glucose response curves and area under the curves during insulin tolerance test (ITT), (I) glucose infusion rate (GIR) during a hyperinsulinemic euglycemic clamp, (J) body mass of the WT and iECKO mice before and after the consumption of a high fat (HF) diet for 8 weeks, (K, L) blood glucose response curves and area under the curves (AUC) during glucose tolerance test of a normal chow (NC) and HF fed mice. Data are shown as mean ± SEM with individual datapoints. N=5-8/group for euglycemic clamps, body mass and tissue mass data; N=16-25/group for all other assays. Independent Student’s t test, paired t tests and RM-ANOVA were performed to assess group differences. *p≤0.03 vs WT; †p≤0.03 vs baseline or NC.

Article Snippet: Protein expression was measured by standard western blot procedures using anti-mouse primary antibodies against Arf6 (rabbit monoclonal; 1:1000; 19kDa; Cell Signaling), β-actin (mouse monoclonal; 1:1000; 40kDa; Abcam), total Akt (pan-Akt; 1:1000; 60 kDa; cell signaling), phosphorylated Akt (s-473 p-Akt; 1:1000; 60 kDa; cell signaling), phosphorylated eNOS; 1:1000; cell signaling).

Techniques: Knock-Out, Clinical Proteomics, Cell Function Assay

Journal: Journal of Nanobiotechnology

Article Title: GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca 2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer

doi: 10.1186/s12951-022-01530-6

Figure Lengend Snippet: Cell-cycle-dependent centrosomal enrichment of GRP75 and its phosphorylation acting on sub-phase distribution. (A) Cyclebase-based predication of expression of GRP75, MPS1, VDAC1 and Arf6 in sub-phases of cell-cycle (dark blue content indexes the mRNA level during cell-cycle). (B) Western blot determined the expression level of those mentioned protein in subcellular fractions of synchronized Skov3. Protein bands were quantified by Image J software and expression ratio (compared to cells without drug-induction, Ctrl, set as 1) in the sub-phase are correspondingly marked below. (C) Schematic GRP75 constructs: wt: wild type. unphospho: unphosphorlation (T62A/S65A). phospho mimic: phosphorlation mimic (T62D/S65D). (D ) Lentivirus-stable-transduced Skov3 and Cos7 cells were built based on GRP75-targeting shRNA (GRP75-KD), or over-expression (OE) with constructs as listed above. GRP75 expression level was analyzed by western blotting. ( E ) Bar graphs show the sub-phase percentage of cell-cycle in GRP75-KD or -OE cells. Data are the average results of three independent experiments

Article Snippet: Rabbit anti-Arf6 Ab (381658) was obtained from Zenbio.

Techniques: Phospho-proteomics, Expressing, Western Blot, Software, Construct, shRNA, Over Expression