mouse anti arf6 mab  (Santa Cruz Biotechnology)

Journal: Nature Communications

Article Title: Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function

doi: 10.1038/s41467-023-43848-1

Figure Lengend Snippet: a , b ARF6 activity (ARF6 GTP) assessed by effector pull-down in HaCaT keratinocytes in the presence or absence of 10 µg/ml CAR or m CAR peptide. a Representative blots of ARF6 activity during time-course. ARF6 GTP: ARF6 detection in GST-GGA3 pull-down eluate. Total ARF6: ARF6 expression in total cell lysate. Tubulin: total cell lysate loading control. b Mean ARF6 activity, relative to total ARF6 ± S.E.M. normalised to 0 min Nil treatment. N = 3–7 independent biological replicate experiments (Nil: 0 min N = 7, 60 min N = 3; CAR: 10–60 min N = 3, 120 min N = 7; m CAR: 30–60 min N = 3, 120 min N = 6). Datapoints represent ARF6 activity per experiment. Two-way ANOVA with Tukey’s multiple comparisons test: Nil 0’ vs CAR 120’ P = 6.457 × 10 −5 ; CAR 120’ vs m CAR 120’ P = 6.732 × 10 −6 ; Nil 60’ vs CAR 60’ P = 4.865 × 10 −5 ; CAR 60’ vs m CAR 60’ P = 0.0063. Holm-Sidak t test: Nil 0’ vs CAR 10’ P = 0.0004. c – e ARF6 activity in HaCaT cells transfected with control siRNA (CTRL KD), human SDC4-targeting siRNA oligo #1 (SDC4 KD #1) or human SDC4-targeting siRNA oligo #2 (SDC4 KD #2), in the presence or absence of 10 µg/ml CAR or m CAR peptide. c Representative blots of ARF6 activity during time-course. ARF6 GTP: ARF6 detection in GST-GGA3 pull-down eluate; total ARF6: ARF6 expression in total cell lysate. d Mean ARF6 activity, relative to total ARF6 ± S.E.M. normalised to Nil treatment. N = 3–5 independent replicate experiments (Ctrl KD: Nil 0 min N = 5; CAR 10 min N = 4, 30–120 min N = 5; mCAR 120 min N = 3. SDC4 KD #1: Nil 0 min N = 3; CAR 10–120 min N = 3; m CAR 120 min N = 3. SDC4 KD #5: Nil 0 min N = 5; CAR 10–120 min N = 5; m CAR 120 min N = 3). Datapoints represent ARF6 activity per experiment. Two-way ANOVA with Tukey’s multiple comparisons test: CTRL KD Nil 0’ vs CAR 120’ P = 0.0299; CTRL KD CAR 120’ vs SDC4 KD #1 CAR 120’ P = 0.0499; CTRL KD CAR 120’ vs SDC4 KD #2 CAR 120’ P = 0.0132. Holm-Sidak t test: CTRL KD Nil 0’ vs CTRL KD CAR 10’ P = 0.0012. Source data are provided in file. e Flow cytometric analysis of cell surface SDC4 in HaCaTs following SDC4 knockdown.

Article Snippet: Following permeabilisation with 0.1% (v/v) Triton X-100 for 10 min and blockade with 2% BSA in PBS - for 1 h, cells were incubated with either 5 μg/ml rabbit anti-ARF6 pAb (#PA1-093, Invitrogen) and 5 μg/ml mouse anti-CYTH2 mAb (10A12; #MA1-061, Pierce), or 10 μg/ml mouse anti-ARF6 mAb (3A-1; #sc-7971, Santa Cruz) and 5 μg/ml rabbit anti-IQSEC1 pAb (#PA5-38019, Invitrogen), in PBS - containing 0.5% (w/v) BSA and 0.05% TritonX-100 for 1 h. Primary antibodies were detected using 1:400 AlexaFluor-488 and −647-conjugated species-specific secondary antibodies and actin was detected using AlexaFluor-594-conjugated phalloidin (1:400; A12381, Invitrogen).

Techniques: Activity Assay, Expressing, Control, Transfection, Knockdown

Journal: Nature Communications

Article Title: Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function

doi: 10.1038/s41467-023-43848-1

Figure Lengend Snippet: Migration of HaCaT keratinocytes on fibronectin in scratch wound assays, in the presence or absence of 10 µg/ml CAR or m CAR peptide. Cells were analysed over 17 h ( a – d ) or 20 h ( e – h ) by time-lapse microscopy. a – d HaCaT cells transfected with control siRNA (CTRL KD), human SDC4-targeting siRNA oligo #1 (SDC4 KD #1) or human SDC4-targeting siRNA oligo #2 (SDC4 KD #2). a Scratch wound closure, ( b ) speed at early phase of migration (Timepoint 0–5 h), ( c ) speed at late phase of migration (Late: Timepoint 12–17 h), and ( d ) representative migration tracks during late phase migration. See also Supplementary Movie and and Supplementary Fig. . Data are representative from one of four independent experiments. Values are means ± S.E.M. All statistical analyses are two-way ANOVA with Tukey’s multiple comparisons test. a n = 5–6 fields of view per condition (Ctrl KD: Nil & CAR n = 6; m CAR n = 5. SDC4 KD #1 & #2: Nil, CAR & m CAR n = 5); CTRL KD Nil vs CAR P = 1.383 × 10 −7 ; CTRL KD CAR vs m CAR P = 2.239 × 10 −6 ; CTRL KD CAR vs SDC4 KD #1 CAR P = 4.341 × 10 −6 ; CTRL KD CAR vs SDC4 KD #2 CAR P = 2.630 × 10 −5 . b , c n = 40–60 cells per condition. b Early phase: CTRL KD Nil vs CAR P = 9.808 × 10 −6 ; CTRL KD CAR vs m CAR P = 1.639 × 10 −9 . (c) Late phase: CTRL KD Nil vs CAR P = 2.520 × 10 −11 ; CTRL KD CAR vs m CAR P = 2.520 × 10 −11 . e – h HaCaT cells transfected with control siRNA (CTRL KD) or human ARF6-targeting siRNA. e Scratch wound closure, ( f ) speed at early phase of migration (Timepoint 0–5 h), ( g ) speed at late phase of migration (Late: Timepoint 15–20 h), and ( e ) representative migration tracks during late phase migration. See also Supplementary Movie and and Supplementary Fig. . Data are representative from one of four independent experiments. Values are means ± S.D. All statistical analyses are two-way ANOVA with Tukey’s multiple comparisons test. e n = 4–6 fields of view per condition (Ctrl KD: Nil n = 4, CAR n = 3; m CAR n = 5. ARF6 KD: Nil, CAR & m CAR n = 6); CTRL KD Nil vs CAR P = 6.312 × 10 −6 ; CTRL KD CAR vs m CAR P = 7.806 × 10 −5 ; CTRL KD CAR vs ARF6 KD CAR P = 1.636 × 10 −6 . f , g n = 49–60 cells per condition (Ctrl KD: Nil & CAR n = 60, m CAR n = 59. ARF6 KD: Nil n = 58, CAR & m CAR n = 49). f Early phase: CTRL KD Nil vs CAR P = 6.82 × 10 −13 ; CTRL KD CAR vs m CAR P = 0.0006; CTRL KD CAR vs ARF6 KD CAR P = 8.897 × 10 −6 ( g ) Late phase: CTRL KD Nil vs CAR P = 4.68 × 10 −13 ; CTRL KD CAR vs m CAR P = 4.68 × 10 −13 ; CTRL KD CAR vs ARF6 KD CAR P = 4.68 × 10 −13 ( a , e ) Each data point represents a single field of view; ( b , c , e , f ) Each data point represents an individual cell. Source data are provided as a file.

Article Snippet: Following permeabilisation with 0.1% (v/v) Triton X-100 for 10 min and blockade with 2% BSA in PBS - for 1 h, cells were incubated with either 5 μg/ml rabbit anti-ARF6 pAb (#PA1-093, Invitrogen) and 5 μg/ml mouse anti-CYTH2 mAb (10A12; #MA1-061, Pierce), or 10 μg/ml mouse anti-ARF6 mAb (3A-1; #sc-7971, Santa Cruz) and 5 μg/ml rabbit anti-IQSEC1 pAb (#PA5-38019, Invitrogen), in PBS - containing 0.5% (w/v) BSA and 0.05% TritonX-100 for 1 h. Primary antibodies were detected using 1:400 AlexaFluor-488 and −647-conjugated species-specific secondary antibodies and actin was detected using AlexaFluor-594-conjugated phalloidin (1:400; A12381, Invitrogen).

Techniques: Migration, Time-lapse Microscopy, Transfection, Control

Journal: Nature Communications

Article Title: Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function

doi: 10.1038/s41467-023-43848-1

Figure Lengend Snippet: a , b Proteomic analysis of ARF regulatory molecules co-immunoprecipitating with human SDC4 (huSDC4) from Syn4WT, Syn4-/-, Syn4Y180E and Syn4Y180L MEFs. a Protein-protein interaction network of molecules in the GO Term “Regulation of ARF Protein Signal Transduction” [GO:0032012]. GEFs: green nodes; GAPs: purple nodes; Blue nodes: proteins not in GO:0032012 (ARF6, ARF1 GTPases and SDC4 bait protein); Edges (grey lines): known protein-protein interactions; Black labels: proteins with reported ARF6 activity modulation properties ; white labels: proteins not reported to modulate ARF6 activity . Red dashed box: proteins co-immunoprecipitating with huSDC4. b Heatmap displaying proteins within GO:0032012 co-immunoprecipitating with huSDC4. Colour-coding indicates enrichment levels (weighted spectral counts). c , d Quantitative analysis of CYTH2/ARF6 co-localisation following CAR peptide stimulation. c Subcellular distribution of CYTH2 (magenta) and ARF6 (green) following 60-min treatment 10 µg/ml CAR, m CAR or vehicle control. Dashed boxes: inset regions. Scale bars: 5 μm (main images); 2 μm (insets). d Pearson’s coefficient of CYTH2 and ARF6 co-localisation ± S.E.M. following 0–120-min treatment with 10 µg/ml CAR or vehicle control. Datapoints represent mean Pearson’s coefficient of CYTH2 and ARF6 co-localisation per image. N = 3 independent replicate experiments with 17–22 images analysed per condition. Kruskal-Wallis test with Dunn’s multiple comparisons test: Nil 0’ vs CAR 60’ P = 7.385 × 10 −5 ; Nil 60’ vs CAR 60’ P = 0.0019; CAR 60’ vs m CAR 60’ P = 3.828 × 10 −6 . e CAR peptide promotes association of CYTH2 with ARF6. Immunoprecipitation of ARF6 following 0, 30 or 60 min CAR or m CAR treatment. immunoprecipitation with rabbit anti-ARF6 (IP: ARF6); or non-immune rabbit IgG (IP: IgG). Immune complex-associated CYTH2 and ARF6 detected by western blot. f , g ARF6 activity in CTRL KD or CYTH2 KD keratinocytes following 120 min in presence or absence of CAR or m CAR. N = 4 Independent biological replicate experiments. f Representative blots of ARF6 GTP (GST-GGA3 pull-down eluate); Total ARF6: ARF6 expression in total cell lysate. Actin detection in TCL acts as a loading control. CYTH2 detection in TCL demonstrates level of siRNA-mediated knockdown. g Mean ARF6 activity relative to total ARF6 ± S.E.M. normalised to Nil treatment in Ctrl KD cells. N = 4 independent replicate experiments. Datapoints represent ARF6 activity in each experiment. Brown–Forsythe and Welch ANOVA test for multiple comparisons assuming non-equal variance: CTRL KD Nil vs CAR P = 0.0027; CTRL KD CAR vs m CAR P = 0.0213. h – k Migration of control (CTRL KD) or human CYTH2 knockdown (CYTH2 KD) HaCaTs in presence or absence 10 µg/ml CAR or m CAR peptide. h Scratch wound closure relative to untreated Ctrl KD cells, ( i ) mean migration speed throughout timelapse, j speed at early migration phase (0–5 h), k speed at late migration phase (12–17 h). Migration data are means ± S.E.M from three independent experiments in triplicate. Statistical analyses are two-way ANOVA with Tukey’s multiple comparisons test: h Scratch wound closure: CTRL KD Nil vs CAR P = 3.331 × 10 −5 ; CTRL KD CAR vs m CAR P = 0.0005; i Total migration speed: CTRL KD Nil vs CAR P = 0.017; CTRL KD CAR vs m CAR ns; k Late phase: CTRL KD Nil vs CAR P = 0.0039; CTRL KD CAR vs m CAR P = 0.0317. Source data are provided as a file.

Article Snippet: Following permeabilisation with 0.1% (v/v) Triton X-100 for 10 min and blockade with 2% BSA in PBS - for 1 h, cells were incubated with either 5 μg/ml rabbit anti-ARF6 pAb (#PA1-093, Invitrogen) and 5 μg/ml mouse anti-CYTH2 mAb (10A12; #MA1-061, Pierce), or 10 μg/ml mouse anti-ARF6 mAb (3A-1; #sc-7971, Santa Cruz) and 5 μg/ml rabbit anti-IQSEC1 pAb (#PA5-38019, Invitrogen), in PBS - containing 0.5% (w/v) BSA and 0.05% TritonX-100 for 1 h. Primary antibodies were detected using 1:400 AlexaFluor-488 and −647-conjugated species-specific secondary antibodies and actin was detected using AlexaFluor-594-conjugated phalloidin (1:400; A12381, Invitrogen).

Techniques: Transduction, Protein-Protein interactions, Activity Assay, Control, Immunoprecipitation, Western Blot, Expressing, Knockdown, Migration

Journal: Nature Communications

Article Title: Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function

doi: 10.1038/s41467-023-43848-1

Figure Lengend Snippet: Schematic diagram highlighting proposed mechanism of action of CAR peptide. Systemically administered CAR peptide associates with the HSPG SDC4, which is restricted to epidermis and blood vessels in mouse skin wounds. CAR induces SDC4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor CYTH2, to promote SDC4-, ARF6- and CYTH2-mediated keratinocyte migration and endogenous re-epithelialisation and wound repair mechanisms.

Article Snippet: Following permeabilisation with 0.1% (v/v) Triton X-100 for 10 min and blockade with 2% BSA in PBS - for 1 h, cells were incubated with either 5 μg/ml rabbit anti-ARF6 pAb (#PA1-093, Invitrogen) and 5 μg/ml mouse anti-CYTH2 mAb (10A12; #MA1-061, Pierce), or 10 μg/ml mouse anti-ARF6 mAb (3A-1; #sc-7971, Santa Cruz) and 5 μg/ml rabbit anti-IQSEC1 pAb (#PA5-38019, Invitrogen), in PBS - containing 0.5% (w/v) BSA and 0.05% TritonX-100 for 1 h. Primary antibodies were detected using 1:400 AlexaFluor-488 and −647-conjugated species-specific secondary antibodies and actin was detected using AlexaFluor-594-conjugated phalloidin (1:400; A12381, Invitrogen).

Techniques: Activation Assay, Migration