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Santa Cruz Biotechnology anti n wasp scbt cat sc 271484
Anti N Wasp Scbt Cat Sc 271484, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology wasp
<t>Hem1-deficiency</t> promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and <t>WASP</t> from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.
Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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wasp - by Bioz Stars, 2024-07
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Images

1) Product Images from "Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption"

Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption

Journal: Scientific Reports

doi: 10.1038/s41598-024-58110-x

Hem1-deficiency promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and WASP from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.
Figure Legend Snippet: Hem1-deficiency promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and WASP from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.

Techniques Used: In Vitro, Osteoclast differentiation Assay, Staining, TRAP Assay, Two Tailed Test, Cell Culture, Lysis, MANN-WHITNEY, Western Blot


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Santa Cruz Biotechnology hpa039490 anti wasp
Hpa039490 Anti Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology rabbit anti n wasp
Rabbit Anti N Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology mouse anti wasp
(a) Enrichment of <t>N-WASP,</t> WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
Mouse Anti Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti wasp/product/Santa Cruz Biotechnology
Average 86 stars, based on 1 article reviews
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Images

1) Product Images from "Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse"

Article Title: Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

Journal: bioRxiv

doi: 10.1101/2023.11.27.568897

(a) Enrichment of N-WASP, WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
Figure Legend Snippet: (a) Enrichment of N-WASP, WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).

Techniques Used: Expressing, Infection, Membrane, Over Expression

(a) Testing the specificity of the WAVE2 polyclonal antibody used in this study. The WAVE2 KD C2C12 cells were generated as described in . After five days of puromycin selection, the surviving cells were mixed with wild-type C2C12 cells and seeded at 30% confluence in GM. After 24 hours, the cells were immunostained with anti-WAVE2 and phalloidin. The WAVE2 KD cells were marked by mCherry expression. Note that the wild-type cells had enriched WAVE2 along the plasma membrane at cell-cell contact sites. The nuclear staining in WAVE2 KD cells is non-specific indicated by western blot . Three experiments were performed with similar results. (b-c) Stills from a time-lapse series showing that N-WASP ( b ) and WIP2 ( c ) colocalize with F-actin along the entire length of straight protrusions. C2C12 cells co-expressing LifeAct-mNG and mScar-N-WASP (or mScar-WIP2) were infected with retrovirus containing MyoD. At 48 hours post MyoD overexpression, the cells were subjected to live imaging (see Supplemental Video 7 and 8). Arrowheads point to randomly selected protrusions. Three experiments for mScar-N-WASP and mScar-WIP2 each were performed with similar results. Scale bars: 30 μm ( a ) and 5 μm ( b and c ).
Figure Legend Snippet: (a) Testing the specificity of the WAVE2 polyclonal antibody used in this study. The WAVE2 KD C2C12 cells were generated as described in . After five days of puromycin selection, the surviving cells were mixed with wild-type C2C12 cells and seeded at 30% confluence in GM. After 24 hours, the cells were immunostained with anti-WAVE2 and phalloidin. The WAVE2 KD cells were marked by mCherry expression. Note that the wild-type cells had enriched WAVE2 along the plasma membrane at cell-cell contact sites. The nuclear staining in WAVE2 KD cells is non-specific indicated by western blot . Three experiments were performed with similar results. (b-c) Stills from a time-lapse series showing that N-WASP ( b ) and WIP2 ( c ) colocalize with F-actin along the entire length of straight protrusions. C2C12 cells co-expressing LifeAct-mNG and mScar-N-WASP (or mScar-WIP2) were infected with retrovirus containing MyoD. At 48 hours post MyoD overexpression, the cells were subjected to live imaging (see Supplemental Video 7 and 8). Arrowheads point to randomly selected protrusions. Three experiments for mScar-N-WASP and mScar-WIP2 each were performed with similar results. Scale bars: 30 μm ( a ) and 5 μm ( b and c ).

Techniques Used: Generated, Selection, Expressing, Membrane, Staining, Western Blot, Infection, Over Expression, Imaging

(a) Still images of cortical protrusions of wild-type, NPF KO and Arp2 KD C2C12 cells at 48 hours post MyoD overexpression. The N-WASP -/- , WIP2 -/- , WAVE2 -/- and Arp2 KD cells expressing LifeAct-mNG were subjected to live imaging as described in (see Supplemental Video 9). Randomly picked straight/stiff protrusions (arrowheads), bendy/soft protrusions (arrows), and lamellipodia (asterisks) are shown. Note the straight/stiff vs. bendy/soft finger-like protrusions projected out of the protruding front of lamellipodia in wild-type vs. N-WASP -/- cells; the absence of lamellipodia in WIP2 -/- , WAVE2 -/- , and Apr2-KD cells; the bendy/soft protrusions in WIP2 -/- cell; decreased number of protrusions in WAVE2 -/- cell; and no protrusions in Arp2 KD cells. (b) Quantification of the number of newly formed straight/stiff protrusion per 10 μm cell boundary per minute for cells with the genotypes shown in ( a ). n = 20-30 cells were measured for each genotype in each experiment and three independent experiments were performed. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. **: p < 0.01. (c-g) Localization of N-WASP, WIP2, WAVE2 and Vasp in individual protrusions of wild-type and mutant MyoD OE C2C12 cells. The wild-type ( c and d ), mNG-N-WASP expressing WAVE2 -/- ( e ), N-WASP -/- ( f ), and mNG-N-WASP expressing WIP2 -/- ( g ) cells were immunostained with anti-WAVE2, phalloidin and/or Vasp at 48 hours post MyoD overexpression. Note that N-WASP and WIP2 (arrowheads) were enriched in the shafts of the invasive protrusions in wild-type and WAVE2 -/- cells, WAVE2 was enriched at the leading edge of the lamellipodia (arrows) including tips of protrusions where VASP was enriched (arrowheads) in N-WASP -/- cells, and that N-WASP, WAVE2 and Vasp (arrowheads) were all enriched at the tips of the protrusions in WIP2 -/- cells. Three independent experiments were performed with similar results. (h) N-WASP is required for generating dynamic finger-like protrusions. At 48 hours post MyoD overexpression, 0.05% DMSO or 5 μM WISK dissolved in DMSO was added to the medium of the LifeAct-mNG expressing C2C12 cells and cells were immediately subjected to live imaging. The invasive protrusions are indicated by arrowheads, and lamellipodia by arrows, respectively (see Supplemental Video 10). Three independent experiments were performed with similar results. Scale bars: 10 μm ( a and h ), 3 μm ( c , e , f and g ) and 5 μm ( d ).
Figure Legend Snippet: (a) Still images of cortical protrusions of wild-type, NPF KO and Arp2 KD C2C12 cells at 48 hours post MyoD overexpression. The N-WASP -/- , WIP2 -/- , WAVE2 -/- and Arp2 KD cells expressing LifeAct-mNG were subjected to live imaging as described in (see Supplemental Video 9). Randomly picked straight/stiff protrusions (arrowheads), bendy/soft protrusions (arrows), and lamellipodia (asterisks) are shown. Note the straight/stiff vs. bendy/soft finger-like protrusions projected out of the protruding front of lamellipodia in wild-type vs. N-WASP -/- cells; the absence of lamellipodia in WIP2 -/- , WAVE2 -/- , and Apr2-KD cells; the bendy/soft protrusions in WIP2 -/- cell; decreased number of protrusions in WAVE2 -/- cell; and no protrusions in Arp2 KD cells. (b) Quantification of the number of newly formed straight/stiff protrusion per 10 μm cell boundary per minute for cells with the genotypes shown in ( a ). n = 20-30 cells were measured for each genotype in each experiment and three independent experiments were performed. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. **: p < 0.01. (c-g) Localization of N-WASP, WIP2, WAVE2 and Vasp in individual protrusions of wild-type and mutant MyoD OE C2C12 cells. The wild-type ( c and d ), mNG-N-WASP expressing WAVE2 -/- ( e ), N-WASP -/- ( f ), and mNG-N-WASP expressing WIP2 -/- ( g ) cells were immunostained with anti-WAVE2, phalloidin and/or Vasp at 48 hours post MyoD overexpression. Note that N-WASP and WIP2 (arrowheads) were enriched in the shafts of the invasive protrusions in wild-type and WAVE2 -/- cells, WAVE2 was enriched at the leading edge of the lamellipodia (arrows) including tips of protrusions where VASP was enriched (arrowheads) in N-WASP -/- cells, and that N-WASP, WAVE2 and Vasp (arrowheads) were all enriched at the tips of the protrusions in WIP2 -/- cells. Three independent experiments were performed with similar results. (h) N-WASP is required for generating dynamic finger-like protrusions. At 48 hours post MyoD overexpression, 0.05% DMSO or 5 μM WISK dissolved in DMSO was added to the medium of the LifeAct-mNG expressing C2C12 cells and cells were immediately subjected to live imaging. The invasive protrusions are indicated by arrowheads, and lamellipodia by arrows, respectively (see Supplemental Video 10). Three independent experiments were performed with similar results. Scale bars: 10 μm ( a and h ), 3 μm ( c , e , f and g ) and 5 μm ( d ).

Techniques Used: Over Expression, Expressing, Imaging, Two Tailed Test, Mutagenesis


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Santa Cruz Biotechnology n wasp
N Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Santa Cruz Biotechnology rabbit anti n wasp
Rabbit Anti N Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti n wasp/product/Santa Cruz Biotechnology
Average 86 stars, based on 1 article reviews
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rabbit anti n wasp - by Bioz Stars, 2024-07
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Santa Cruz Biotechnology anti wasp
Anti Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti wasp/product/Santa Cruz Biotechnology
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
anti wasp - by Bioz Stars, 2024-07
86/100 stars

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Santa Cruz Biotechnology anti n wasp
Anti N Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti n wasp/product/Santa Cruz Biotechnology
Average 86 stars, based on 1 article reviews
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anti n wasp - by Bioz Stars, 2024-07
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antibody against neural wiskott aldrich syndrome protein n wasp  (Santa Cruz Biotechnology)

 
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    Structured Review

    Santa Cruz Biotechnology antibody against neural wiskott aldrich syndrome protein n wasp
    Antibody Against Neural Wiskott Aldrich Syndrome Protein N Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology anti n wasp scbt cat sc 271484
    Anti N Wasp Scbt Cat Sc 271484, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti n wasp scbt cat sc 271484/product/Santa Cruz Biotechnology
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    <t>Hem1-deficiency</t> promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and <t>WASP</t> from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.
    Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    <t>Hem1-deficiency</t> promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and <t>WASP</t> from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.
    Hpa039490 Anti Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit anti n wasp
    <t>Hem1-deficiency</t> promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and <t>WASP</t> from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.
    Rabbit Anti N Wasp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (a) Enrichment of <t>N-WASP,</t> WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
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    (a) Enrichment of <t>N-WASP,</t> WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
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    (a) Enrichment of <t>N-WASP,</t> WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
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    (a) Enrichment of <t>N-WASP,</t> WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
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    (a) Enrichment of <t>N-WASP,</t> WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).
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    Image Search Results


    Hem1-deficiency promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and WASP from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.

    Journal: Scientific Reports

    Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption

    doi: 10.1038/s41598-024-58110-x

    Figure Lengend Snippet: Hem1-deficiency promotes osteoclastogenesis but inhibits resorptive capacity. ( A ) In-vitro osteoclast differentiation assay of WT and Hem1 -/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. ( B ) Quantification of TRAP-assay of WT and Hem1 -/- shown in ( A ). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. ( C ) Representative images of WT and Hem1 -/- osteoclasts cultured on calcium phosphate (CaPO 4 2- ; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. ( D ) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. ( E ) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Immunoblotting of osteoclast markers from WT and Hem1 -/- osteoclast lysates, representative images from n = 4 independent experiments. ( G ) Immunoblotting of Hem1, WAVE2 and WASP from WT and Hem1 -/- osteoclast lysates, representative images from n = 5 independent experiments.

    Article Snippet: Specific antibodies against the following proteins were detected by immunoblotting: cathepsin K (abcam, ab 19,027, 1:1000), integrin alpha-v (abcam, ab179475,1:1000), integrin beta-3 (Cell Signaling #4702,1:1000), DC-STAMP (sc-98769, 1:200), NFATc1 (sc-7294; both from Santa Cruz), Hem1 (Novus Bio, NBP2-13,643, 1:1000), WASP (Santa Cruz, sc-13139, 1:1000) and WAVE2 (Cell Signaling, #3659, 1:1000).

    Techniques: In Vitro, Osteoclast differentiation Assay, Staining, TRAP Assay, Two Tailed Test, Cell Culture, Lysis, MANN-WHITNEY, Western Blot

    (a) Enrichment of N-WASP, WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).

    Journal: bioRxiv

    Article Title: Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

    doi: 10.1101/2023.11.27.568897

    Figure Lengend Snippet: (a) Enrichment of N-WASP, WIP2 and Arp2 within the F-actin structure at the fusogenic synapse of C2C12 cells. Stable C2C12 cell lines co-expressing LifeAct-mNeongreen (mNG) and mScar-N-WASP (or mScar-WIP2, Arp2-mScar, mScar-WAVE2) were infected with retroviruses containing MyoD and imaged as described in . Note the co-enrichment of F-actin and N-WASP, WIP2 and Arp2, but not WAVE2, at the fusogenic synapses (indicated by arrowheads) (see Supplemental Video 6). At least 15 fusogenic synapse were imaged for each target protein with similar results. (b) Complementary localization of WAVE2 and NWASP/WIP2 in the invasive structure. C2C12 cells co-expressing mNG-WIP2 and mScar-N-WASP were infected by retroviruses containing MyoD. After 48 hours, the cells were immunostained with anti-WAVE2. Note that WAVE2 was enriched at the leading edge of the invasive front as a thin layer underneath the plasma membrane, whereas N-WASP/WIP2 colocalized with F-actin within the invasive structure. n = 60 cells were imaged with similar results. (c-f) Invasive protrusions and filopodia are molecularly distinct. After 48 hours of MyoD overexpression, mNG-N-WASP or mNG-WIP2 expressing C2C12 cells were immunostained with anti-WAVE2, anti-Vasp and phalloidin. Note that the thick Vasp − protrusions ( c and e ) had enriched N-WASP/WIP2 in the shafts, whereas the Vasp + filopodia ( d and f ) contained Vasp and N-WASP/WIP2 enrichment at the tips. n = 75 protrusions of each type in at least 30 cells were imaged with similar results. (g) Vasp is not enriched at the tips of the invasive protrusions at the myoblast contact sites. After 48 hours of MyoD overexpression, wild-type C2C12 cells were immunostained with anti-Vasp and phalloidin. Note that Vasp was not enriched at the tips of the invasive protrusions. Scale bars, 5 μm ( a ), 3 μm ( b , e , f and g ) and 2 μm ( c and d ).

    Article Snippet: The following primary antibodies were used: mouse anti-WASP (1:1000; Santa Cruz; 4860), rabbit anti-N-WASP (1:1000; Cell Signaling Technologies; 4848), mouse anti-WIP1 (1:1000, Santa Cruz; sc-271113), rabbit anti-WIP2 (1:1000; ThermoFisher Scientific; PA5-55086), mouse anti-WAVE1 (1:1000; Santa Cruz; sc-271507), rabbit anti-WAVE2 (1:1000; Cell Signaling Technologies; 3659), rabbit anti-WAVE3 (1:1000; Cell Signaling Technologies; 2806), mouse anti-Arp2 (1:1000; Santa Cruz; sc-166103), mouse anti-Arp3 (1:1000; Santa Cruz; sc-48344), mouse anti-MyoG (1:30; DSHB; F5D), mouse anti-MHC (1:200; DSHB; MF20), rabbit anti-ABI1 (1:1000; Cell Signaling Technologies; 39444), rabbit anti-Cyfip1(1:1000; Cell Signaling Technologies; 81221), mouse anti-mNeongreen (Proteintech; 32F6), mouse anti-actin (Sigma: A4700), sheep anti-ESGP (1:1000; R&D Systems; AF4580) and rabbit anti-β-Tubulin (1:1000; Cell Signaling Technologies; 2146).

    Techniques: Expressing, Infection, Membrane, Over Expression

    (a) Testing the specificity of the WAVE2 polyclonal antibody used in this study. The WAVE2 KD C2C12 cells were generated as described in . After five days of puromycin selection, the surviving cells were mixed with wild-type C2C12 cells and seeded at 30% confluence in GM. After 24 hours, the cells were immunostained with anti-WAVE2 and phalloidin. The WAVE2 KD cells were marked by mCherry expression. Note that the wild-type cells had enriched WAVE2 along the plasma membrane at cell-cell contact sites. The nuclear staining in WAVE2 KD cells is non-specific indicated by western blot . Three experiments were performed with similar results. (b-c) Stills from a time-lapse series showing that N-WASP ( b ) and WIP2 ( c ) colocalize with F-actin along the entire length of straight protrusions. C2C12 cells co-expressing LifeAct-mNG and mScar-N-WASP (or mScar-WIP2) were infected with retrovirus containing MyoD. At 48 hours post MyoD overexpression, the cells were subjected to live imaging (see Supplemental Video 7 and 8). Arrowheads point to randomly selected protrusions. Three experiments for mScar-N-WASP and mScar-WIP2 each were performed with similar results. Scale bars: 30 μm ( a ) and 5 μm ( b and c ).

    Journal: bioRxiv

    Article Title: Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

    doi: 10.1101/2023.11.27.568897

    Figure Lengend Snippet: (a) Testing the specificity of the WAVE2 polyclonal antibody used in this study. The WAVE2 KD C2C12 cells were generated as described in . After five days of puromycin selection, the surviving cells were mixed with wild-type C2C12 cells and seeded at 30% confluence in GM. After 24 hours, the cells were immunostained with anti-WAVE2 and phalloidin. The WAVE2 KD cells were marked by mCherry expression. Note that the wild-type cells had enriched WAVE2 along the plasma membrane at cell-cell contact sites. The nuclear staining in WAVE2 KD cells is non-specific indicated by western blot . Three experiments were performed with similar results. (b-c) Stills from a time-lapse series showing that N-WASP ( b ) and WIP2 ( c ) colocalize with F-actin along the entire length of straight protrusions. C2C12 cells co-expressing LifeAct-mNG and mScar-N-WASP (or mScar-WIP2) were infected with retrovirus containing MyoD. At 48 hours post MyoD overexpression, the cells were subjected to live imaging (see Supplemental Video 7 and 8). Arrowheads point to randomly selected protrusions. Three experiments for mScar-N-WASP and mScar-WIP2 each were performed with similar results. Scale bars: 30 μm ( a ) and 5 μm ( b and c ).

    Article Snippet: The following primary antibodies were used: mouse anti-WASP (1:1000; Santa Cruz; 4860), rabbit anti-N-WASP (1:1000; Cell Signaling Technologies; 4848), mouse anti-WIP1 (1:1000, Santa Cruz; sc-271113), rabbit anti-WIP2 (1:1000; ThermoFisher Scientific; PA5-55086), mouse anti-WAVE1 (1:1000; Santa Cruz; sc-271507), rabbit anti-WAVE2 (1:1000; Cell Signaling Technologies; 3659), rabbit anti-WAVE3 (1:1000; Cell Signaling Technologies; 2806), mouse anti-Arp2 (1:1000; Santa Cruz; sc-166103), mouse anti-Arp3 (1:1000; Santa Cruz; sc-48344), mouse anti-MyoG (1:30; DSHB; F5D), mouse anti-MHC (1:200; DSHB; MF20), rabbit anti-ABI1 (1:1000; Cell Signaling Technologies; 39444), rabbit anti-Cyfip1(1:1000; Cell Signaling Technologies; 81221), mouse anti-mNeongreen (Proteintech; 32F6), mouse anti-actin (Sigma: A4700), sheep anti-ESGP (1:1000; R&D Systems; AF4580) and rabbit anti-β-Tubulin (1:1000; Cell Signaling Technologies; 2146).

    Techniques: Generated, Selection, Expressing, Membrane, Staining, Western Blot, Infection, Over Expression, Imaging

    (a) Still images of cortical protrusions of wild-type, NPF KO and Arp2 KD C2C12 cells at 48 hours post MyoD overexpression. The N-WASP -/- , WIP2 -/- , WAVE2 -/- and Arp2 KD cells expressing LifeAct-mNG were subjected to live imaging as described in (see Supplemental Video 9). Randomly picked straight/stiff protrusions (arrowheads), bendy/soft protrusions (arrows), and lamellipodia (asterisks) are shown. Note the straight/stiff vs. bendy/soft finger-like protrusions projected out of the protruding front of lamellipodia in wild-type vs. N-WASP -/- cells; the absence of lamellipodia in WIP2 -/- , WAVE2 -/- , and Apr2-KD cells; the bendy/soft protrusions in WIP2 -/- cell; decreased number of protrusions in WAVE2 -/- cell; and no protrusions in Arp2 KD cells. (b) Quantification of the number of newly formed straight/stiff protrusion per 10 μm cell boundary per minute for cells with the genotypes shown in ( a ). n = 20-30 cells were measured for each genotype in each experiment and three independent experiments were performed. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. **: p < 0.01. (c-g) Localization of N-WASP, WIP2, WAVE2 and Vasp in individual protrusions of wild-type and mutant MyoD OE C2C12 cells. The wild-type ( c and d ), mNG-N-WASP expressing WAVE2 -/- ( e ), N-WASP -/- ( f ), and mNG-N-WASP expressing WIP2 -/- ( g ) cells were immunostained with anti-WAVE2, phalloidin and/or Vasp at 48 hours post MyoD overexpression. Note that N-WASP and WIP2 (arrowheads) were enriched in the shafts of the invasive protrusions in wild-type and WAVE2 -/- cells, WAVE2 was enriched at the leading edge of the lamellipodia (arrows) including tips of protrusions where VASP was enriched (arrowheads) in N-WASP -/- cells, and that N-WASP, WAVE2 and Vasp (arrowheads) were all enriched at the tips of the protrusions in WIP2 -/- cells. Three independent experiments were performed with similar results. (h) N-WASP is required for generating dynamic finger-like protrusions. At 48 hours post MyoD overexpression, 0.05% DMSO or 5 μM WISK dissolved in DMSO was added to the medium of the LifeAct-mNG expressing C2C12 cells and cells were immediately subjected to live imaging. The invasive protrusions are indicated by arrowheads, and lamellipodia by arrows, respectively (see Supplemental Video 10). Three independent experiments were performed with similar results. Scale bars: 10 μm ( a and h ), 3 μm ( c , e , f and g ) and 5 μm ( d ).

    Journal: bioRxiv

    Article Title: Molecular Regulation of Invasive Protrusion Formation at the Mammalian Fusogenic Synapse

    doi: 10.1101/2023.11.27.568897

    Figure Lengend Snippet: (a) Still images of cortical protrusions of wild-type, NPF KO and Arp2 KD C2C12 cells at 48 hours post MyoD overexpression. The N-WASP -/- , WIP2 -/- , WAVE2 -/- and Arp2 KD cells expressing LifeAct-mNG were subjected to live imaging as described in (see Supplemental Video 9). Randomly picked straight/stiff protrusions (arrowheads), bendy/soft protrusions (arrows), and lamellipodia (asterisks) are shown. Note the straight/stiff vs. bendy/soft finger-like protrusions projected out of the protruding front of lamellipodia in wild-type vs. N-WASP -/- cells; the absence of lamellipodia in WIP2 -/- , WAVE2 -/- , and Apr2-KD cells; the bendy/soft protrusions in WIP2 -/- cell; decreased number of protrusions in WAVE2 -/- cell; and no protrusions in Arp2 KD cells. (b) Quantification of the number of newly formed straight/stiff protrusion per 10 μm cell boundary per minute for cells with the genotypes shown in ( a ). n = 20-30 cells were measured for each genotype in each experiment and three independent experiments were performed. Mean ± s.d. values are shown in the bar graph, and significance was determined by two-tailed student’s t-test. **: p < 0.01. (c-g) Localization of N-WASP, WIP2, WAVE2 and Vasp in individual protrusions of wild-type and mutant MyoD OE C2C12 cells. The wild-type ( c and d ), mNG-N-WASP expressing WAVE2 -/- ( e ), N-WASP -/- ( f ), and mNG-N-WASP expressing WIP2 -/- ( g ) cells were immunostained with anti-WAVE2, phalloidin and/or Vasp at 48 hours post MyoD overexpression. Note that N-WASP and WIP2 (arrowheads) were enriched in the shafts of the invasive protrusions in wild-type and WAVE2 -/- cells, WAVE2 was enriched at the leading edge of the lamellipodia (arrows) including tips of protrusions where VASP was enriched (arrowheads) in N-WASP -/- cells, and that N-WASP, WAVE2 and Vasp (arrowheads) were all enriched at the tips of the protrusions in WIP2 -/- cells. Three independent experiments were performed with similar results. (h) N-WASP is required for generating dynamic finger-like protrusions. At 48 hours post MyoD overexpression, 0.05% DMSO or 5 μM WISK dissolved in DMSO was added to the medium of the LifeAct-mNG expressing C2C12 cells and cells were immediately subjected to live imaging. The invasive protrusions are indicated by arrowheads, and lamellipodia by arrows, respectively (see Supplemental Video 10). Three independent experiments were performed with similar results. Scale bars: 10 μm ( a and h ), 3 μm ( c , e , f and g ) and 5 μm ( d ).

    Article Snippet: The following primary antibodies were used: mouse anti-WASP (1:1000; Santa Cruz; 4860), rabbit anti-N-WASP (1:1000; Cell Signaling Technologies; 4848), mouse anti-WIP1 (1:1000, Santa Cruz; sc-271113), rabbit anti-WIP2 (1:1000; ThermoFisher Scientific; PA5-55086), mouse anti-WAVE1 (1:1000; Santa Cruz; sc-271507), rabbit anti-WAVE2 (1:1000; Cell Signaling Technologies; 3659), rabbit anti-WAVE3 (1:1000; Cell Signaling Technologies; 2806), mouse anti-Arp2 (1:1000; Santa Cruz; sc-166103), mouse anti-Arp3 (1:1000; Santa Cruz; sc-48344), mouse anti-MyoG (1:30; DSHB; F5D), mouse anti-MHC (1:200; DSHB; MF20), rabbit anti-ABI1 (1:1000; Cell Signaling Technologies; 39444), rabbit anti-Cyfip1(1:1000; Cell Signaling Technologies; 81221), mouse anti-mNeongreen (Proteintech; 32F6), mouse anti-actin (Sigma: A4700), sheep anti-ESGP (1:1000; R&D Systems; AF4580) and rabbit anti-β-Tubulin (1:1000; Cell Signaling Technologies; 2146).

    Techniques: Over Expression, Expressing, Imaging, Two Tailed Test, Mutagenesis