rabbit anti mouse cd29  (Bio-Rad)

Journal: bioRxiv

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity

doi: 10.1101/2023.02.20.529299

Figure Lengend Snippet: β1-integrin is a SNX17 cargo in neurons and plays a role in dendritic spine density. (A) DIV11 rat cortical neurons were infected with lentiviruses carrying scramble or SNX17 shRNAs, and the surface levels of β1-integrin were determined at DIV17 using a surface biotinylation assay. SNX17 knockdown was validated by western blotting of the lysate, and GAPDH was used as a loading control. (B) The levels of surface β1-integrin protein were quantified and normalized to total β1-integrin levels (lysate). Data are expressed as percentage of ctrl-shRNA (ctrl-shRNA: 100%, SNX17-shRNA: 57.630 ± 3.058%). N=4 independent experiments. Statistical significance was determined using unpaired two-tailed Student’s t-test, ****p<0.001. Error bars are SEM. (C) Representative confocal images of surface β1-integrin levels of DIV17 hippocampal neurons that were infected at DIV11 with lentiviruses carrying either ctrl-shRNA or SNX17-shRNA. Neurons were treated in the presence or absence of cLTP and live labeled with an anti-surface ß1-integrin antibody for 15 min, followed by fixation and immunostaining for MAP2. Scale bar, 5 µm. (D) The intensity of ß1-integrin in the first 50 µm of secondary dendrites was quantified, and values were normalized to crtl-shRNA. ctrl-shRNA: 1.000 ± 0.038, N=32 neurons; ctrl-shRNA cLTP: 1.139 ± 0.039, N=29 neurons; SNX17-shRNA: 0.839 ± 0.040, N=28 neurons; SNX17-shRNA cLTP: 0.786 ± 0.035, N=28 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, *p<0.05. Error bars are SEM. (E) Validation of a shRNA clone (V2LMM_39157, Horizon Discovery) to knock-down rat ITGB1. pGIPZ scrambled non-target (RHS4346, Horizon Discovery) was used as a control. HEK293 cells stably expressing the tet repressor (TR-HEK293) were either transfected with control-shRNA or ITGB1-shRNA in the absence or presence of eGFP or ITGB1-GFP, as indicated. 5-days post-infection, cells were treated with 1 μg/ml of doxycycline to promote the expression of eGFP or ITGB1-GFP. 24 hours later, extracts were generated and analyzed by western blot. (F) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or ITGB1-shRNA. Scale bar, 5 µm. treated with either β1-integrin blocking or iso type control antibodies 24 hours before fixation. Scale bar, 5 µm. (G) The numbers of dendritic spines in the first 30 μm of secondary dendrites were quantified. ctrl-shRNA: 0.705 ± 0.044, N=31 neurons; ITGB1-shRNA: 0.505 ± 0.043, N=33 neurons. Statistical significance was determined using unpaired two-tailed Student’s t-test, **p<0.01. Error bars are SEM. (H) Hippocampal neurons were transfected at DIV12 with eGFP (filler) and either ctrl-shRNA or SNX17-shRNA. Neurons were treated with either β1-integrin blocking or isotype control antibodies 24 hours before fixation at DIV16. The number of dendritic spines in the first 30 μm of secondary dendrites was quantified. ctrl-shRNA + isotype ctrl: 0.689 ± 0.030, N=26 neurons; ctrl-shRNA +β1-integrin blocking: 0.451 ± 0.021, N=27 neurons; SNX17-shRNA + isotype ctrl: 0.385 ± 0.026, N=28 neurons; SNX17-shRNA +β1-integrin blocking: 0.387 ± 0.020, N=26 neurons,. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test, ****p<0.001. Error bars are SEM

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1000, HPA043867, Atlas Antibodies), SNX17 mouse mAb, VPS35L rabbit pAb (1:1000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1000, 11938-1-AP, Proteintech), GFP rabbit mAb (1:1000, Ab32146, Abcam), mouse/rabbit Integrin β1 goat pAb (1:1000, AF2405, R&D Systems) and GAPDH rabbit mAb (1:2000, 2118, Cell Signaling).

Techniques: Infection, Surface Biotinylation Assay, Knockdown, Western Blot, Control, shRNA, Two Tailed Test, Labeling, Immunostaining, Biomarker Discovery, Stable Transfection, Expressing, Transfection, Generated, Blocking Assay

Journal: bioRxiv

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity

doi: 10.1101/2023.02.20.529299

Figure Lengend Snippet: β1-integrin has roles in structural and functional plasticity during cLTP. (A) Diagram of experiment. DIV16-18 hippocampal neurons were treated with either β1-integrin blocking or isotype control antibodies for 30 min, followed by a 5 min cLTP stimulus. mEPSCs were recorded during the first 30 min after cLTP (cLTP<30) or from 30 to 90 min after cLTP (cLTP>30). (B) Examples of mEPSC recordings of neurons that were treated with isotype ctrl or β1-integrin blocking antibodies. Recordings were performed in the absence of cLTP (baseline), during the first 30 min after cLTP or from 30 to 90 min after cLTP. (C) Quantification of mEPSC amplitude. Isotype ctrl baseline: 12.240 ± 0.570, N=19 neurons; isotype ctrl cLTP<30min: 16.790 ± 1.106, N=9 neurons; isotype ctrl cLTP>30min: 16.900 ± 0.954, N=10 neurons; β1-integrin blocking baseline: 11.570 ± 0.373, N=9 neurons; β1-integrin blocking cLTP<30 min: 18.110 ± 1.247, N=6 neurons; β1-integrin blocking neurons cLTP>30 min: 13.170 ± 0.458, N=13 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ****p<0.001. Error bars are SEM. (D) Quantification of mEPSC frequency. Isotype ctrl baseline: 1.191 ± 0.227, N=19 neurons; isotype ctrl cLTP<30min: 3.333 ± 0.800, N=9 neurons; isotype ctrl cLTP>30min: 3.110 ± 0.740, N=10 neurons; β1-integrin blocking baseline: 1.167 ± 0.296, N=9 neurons; β1-integrin blocking cLTP<30 min: 3.952 ± 1.214, N=6 neurons; β1-integrin blocking neurons cLTP>30 min: 1.096 ± 0.302, N=13 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, **p<0.01. Error bars are SEM. (E) Representative confocal images of dendritic spines in DIV16 hippocampal neurons transfected with eGFP (filler) at DIV12. Neurons were treated with ß1-integrin blocking or isotype control antibodies for 30 min, followed by a 5-min cLTP stimulus in the presence of antibodies where indicated. Neurons were further incubated in the presence of antibodies for 50 min before fixation. Scale bar, 5 µm. (F) The maximum width for each spine was quantified, and the average size of the dendritic spines in the first 30 μm of secondary dendrites was calculated. Isotype ctrl: 0.626 ± 0.016, N=28 neurons; isotype ctrl with cLTP: 0.717 ± 0.018, N=25 neurons; β1-integrin blocking: 0.621 ± 0.015, N=31 neurons; β1-integrin blocking with cLTP: 0.628 ± 0.010, N=32 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test, ***p<0.005. Error bars are SEM. (G)Quantification of dendritic spine density (spines/μm). Isotype ctrl: 1.044 ± 0.048, N=28 neurons; isotype ctrl with cLTP: 0.977 ± 0.050, N=25 neurons; β1-integrin blocking: 0.931 ± 0.054, N=31 neurons; β1-integrin blocking with cLTP: 0.931 ± 0.039, N=32 neurons. 3 independent experiments. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Error bars are SEM.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1000, HPA043867, Atlas Antibodies), SNX17 mouse mAb, VPS35L rabbit pAb (1:1000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1000, 11938-1-AP, Proteintech), GFP rabbit mAb (1:1000, Ab32146, Abcam), mouse/rabbit Integrin β1 goat pAb (1:1000, AF2405, R&D Systems) and GAPDH rabbit mAb (1:2000, 2118, Cell Signaling).

Techniques: Functional Assay, Blocking Assay, Control, Transfection, Incubation

Journal: bioRxiv

Article Title: Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity

doi: 10.1101/2023.02.20.529299

Figure Lengend Snippet: Model of SNX17-mediated modulation of synaptic structure and function. The SNX17-Retriever pathway is required for dendritic spine maintenance and for the cLTP-dependent increase in dendritic spine size. Glycine-mediated cLTP (1) stimulates calcium entry through the NMDA receptor, which activates the CaMKII pathway (2). CaMKII activation is necessary and sufficient to promote the recruitment of SNX17 and the Retriever complex to dendritic spines (3), and activates the recycling of β1-integrin from endosomes to the plasma membrane (4). The surface levels of β1-integrin increase during cLTP and promote dendritic spine growth (5). Endosomal PI(3)P increases upon cLTP and may help with the recruitment of SNX17 to synapses. Created with BioRender.com.

Article Snippet: Primary antibodies used included SNX17 rabbit pAb (1:1000, HPA043867, Atlas Antibodies), SNX17 mouse mAb, VPS35L rabbit pAb (1:1000, Daniel D. Billadeau), COMMD1 rabbit pAb (1:1000, 11938-1-AP, Proteintech), GFP rabbit mAb (1:1000, Ab32146, Abcam), mouse/rabbit Integrin β1 goat pAb (1:1000, AF2405, R&D Systems) and GAPDH rabbit mAb (1:2000, 2118, Cell Signaling).

Techniques: Activation Assay, Clinical Proteomics, Membrane

Journal: Journal of Materials Science. Materials in Medicine

Article Title: Adipose-derived mesenchymal stem cell seeded Atelocollagen scaffolds for cardiac tissue engineering

doi: 10.1007/s10856-020-06425-2

Figure Lengend Snippet: Expression of CD29 and CD45 on the surface of ADMSCs. a Control group; b CD29-FITC; c CD45-FITC

Article Snippet: The third-generation ADMSCs were collected at a density of 10 6 /ml and incubated with rabbit anti-mouse CD29-FITC antibody (1:500, BD Company, USA) and CD45-FITC antibody (1:500, BD Company, USA) at 4 °C for 60 min.

Techniques: Expressing

Journal: Journal of Neuroinflammation

Article Title: Antinociceptive effect of intrathecal injection of miR-9-5p modified mouse bone marrow mesenchymal stem cells on a mouse model of bone cancer pain

doi: 10.1186/s12974-020-01765-w

Figure Lengend Snippet: Analysis of cell surface marker expressions by flow cytometry after transfection (%, mean ± SD)

Article Snippet: Phycoerythrin-marked rabbit anti-mouse CD29 or CD34 (Serotec, Ltd., UK) and fluorescein isothiocyanate-linked anti-mouse CD44 or CD45 antibody (Serotec, Ltd., UK) were used to stain cell surface markers which subsequently were evaluated using fluorescence-activated cell sorting with flow cytometry.

Techniques: Marker, Flow Cytometry, Transfection

Journal: bioRxiv

Article Title: Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis

doi: 10.1101/2020.01.06.892497

Figure Lengend Snippet: a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb CD29) expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P<0.0001 (Mann-Whitney test, two-sided). f, Brightfield micrographs of tumoroids after 72 h of 3D collagen invasion (left panel) and morphology-based single-cell subtypes (right panel) for indicated conditions in the presence or absence of β1 integrin-activating mAb 9EG7. Insets, morphologies of migrating single-cells. Data show the experimental means ± s.d. (5 tumoroids/condition each experiment, n=3). **** P<0.0001, *** P=0.0008, ** P<0.006, * P<0.03 (two-way ANOVA). Abbreviations: E, elongated; P pseudopodal-amoeboid; B, blebbing-amoeboid; N, normoxia; H, hypoxia; V, vehicle (DMSO); D, DMOG.

Article Snippet: The following antibodies and dyes were used: rabbit polyclonal anti-HIF1α (1:1000 WB, Novus Biologicals, 100-479), rabbit polyclonal anti–calpain-2 large subunit (1:1000 WB, Cell Signaling, 2539), chicken polyclonal anti-β-actin (1:1000 WB, Abcam, 13822), rabbit polyclonal anti-β-actin (1:2000 non-reducing WB in 4T1, Cell Signaling, 4967), rabbit polyclonal anti–talin-1 (clone 8d4; 1:200 WB, Sigma-Aldrich, T3287), mouse monoclonal anti-human CD29 (activated β1-integrin, clone HUTS-4; 1:500 WB, MerckMillipore, 2079Z), mouse monoclonal anti-human CD29 (clone 4B4; 1:1000 WB, 0.5-10 μg/ml functional studies, Beckman Coulter, 6603113), rat monoclonal anti-mouse CD29 (primed β1-integrin, clone 9EG7; 1:500 WB, 1:25 FC, 1:50 IF, BD Biosciences, 553715), rabbit monoclonal anti-mouse CD29 (clone EP1041Y; 1:1000 WB, Millipore, 04-1109), rat IgG2a isotype control (Clone R5-95; 1:25 FC, BD Biosciences, 553927), FITC-conjugated Armenian hamster anti-CD29 (clone HMβ1-1; 1:100 IF, 1:50 FC, Biolegend, 102206), FITC-conjugated Armenian hamster IgG (CloneHTK888; 1:20 FC, Biolegend, 400906), rabbit anti-cleaved caspase-3 (1:200 IF, Cell Signaling, 9664), mouse anti-human CD29 (clone TS2/16; 20 μg/ml, BioLegend, 303010), rabbit-on-rodent HRP-polymer (IHC, Biocare Medical, RMR622), rabbit monoclonal anti-cytokeratin 8 (clone EP1628Y; 1:250 IHC, Abcam, 53280), secondary goat anti-rabbit/mouse/chicken antibodies conjugated to horseradish peroxidase (1:10,000 WB, Jackson, 211-032-171/115-0450174/ 103-035-155), secondary goat anti-rat IgG AF488 (1:200 FC, Thermo Fisher Scientific, 11006), secondary goat anti-rat IgG AF647 (1:200 IF, Thermo Fisher Scientific, A21247), secondary goat anti-rabbit IgG AF633 (1:200 IF, Thermo Fisher Scientific, A21071), phalloidin AF488/546/568/633 (1:100 IF, Thermo Fisher Scientific, A12379/A22283/A12380/A22284), DAPI (1:500 IF, Thermo Fisher Scientific, D21490).

Techniques: In Silico, Migration, Western Blot, Cell Isolation, Adhesive, Expressing, Flow Cytometry, Control, MANN-WHITNEY

Journal: Cytotechnology

Article Title: Isolation and characterization of ovine umbilical cord-derived mesenchymal stem cells

doi: 10.1007/s10616-018-0284-7

Figure Lengend Snippet: Immunofluorescence analysis of ovine UC-MSCs surface markers. a, c, e, g, i, k Control group which only cultured in GM and not stained with antibodies. Counterstaining of nuclei was carried out by DAPI (blue), b stained with CD29 antibody, d stained with CD13 antibody, f stained with CD44 antibody, h stained with CD45 antibody, j stained with CD90 antibody, l stained with CD106. Counterstaining of nuclei was carried out by DAPI (blue). All representative samples are shown at × 100 magnification

Article Snippet: After 24 h, ovine UC-MSCs were fixed with 4% formaldehyde for 30 min, then incubated with rabbit anti-mouse CD29, CD13, CD44, CD45, CD90 and CD106 primary antibodies (all purchased from Boster Biological Technology, Wuhan, China).

Techniques: Immunofluorescence, Control, Cell Culture, Staining