vps34 Search Results


vps34in1  (Selleck Chemicals)
94
Selleck Chemicals vps34in1
Vps34in1, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc12656530-37-4-6?v=Selleck+Chemicals
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anti vps34 antibodies  (Novus Biologicals)
90
Novus Biologicals anti vps34 antibodies
Anti Vps34 Antibodies, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/10__1038_slash_npre__2010__5250__1-62-3-8?v=Novus+Biologicals
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vps34  (Novus Biologicals)
93
Novus Biologicals vps34
Vps34, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/10__1523_slash_jneurosci__5156___10__2010-49-38-41?v=Novus+Biologicals
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vps34  (Selleck Chemicals)
93
Selleck Chemicals vps34
Vps34, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/bio_rxiv__2025__06__12__659257-119-17-21?v=Selleck+Chemicals
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human vps34  (OriGene)
90
OriGene human vps34
Primers used for cloning.
Human Vps34, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc04417027-53-3-10?v=OriGene
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human vps34 - by Bioz Stars, 2026-07
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vps34 proteintech  (Proteintech)
93
Proteintech vps34 proteintech
Primers used for cloning.
Vps34 Proteintech, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc09830432__thnov13p0810s1-42-89-90?v=Proteintech
Average 93 stars, based on 1 article reviews
vps34 proteintech - by Bioz Stars, 2026-07
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pik3c3 cdna  (OriGene)
90
OriGene pik3c3 cdna
Primers used for cloning.
Pik3c3 Cdna, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc05980348-156-0-10?v=OriGene
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vps34  (Bio-Rad)
90
Bio-Rad vps34
( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), <t>Flag-VPS34</t> ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.
Vps34, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc08654373-244-42-68?v=Bio-Rad
Average 90 stars, based on 1 article reviews
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recombinant pik 3c3  (Sino Biological)
90
Sino Biological recombinant pik 3c3
( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), <t>Flag-VPS34</t> ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.
Recombinant Pik 3c3, supplied by Sino Biological, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/merrill_nathan_michael__2017__pi3k_c2a_insights_into_autophagy_and_endocytosis-1655-0-8?v=Sino+Biological
Average 90 stars, based on 1 article reviews
recombinant pik 3c3 - by Bioz Stars, 2026-07
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anti vps34 pik3c3  (Novus Biologicals)
90
Novus Biologicals anti vps34 pik3c3
( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), <t>Flag-VPS34</t> ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.
Anti Vps34 Pik3c3, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc03124552-91-90-100?v=Novus+Biologicals
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vps34 3 × flag  (Addgene inc)
93
Addgene inc vps34 3 × flag
( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), <t>Flag-VPS34</t> ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.
Vps34 3 × Flag, supplied by Addgene inc, 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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vps34 3 × flag - by Bioz Stars, 2026-07
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ha vps34  (Addgene inc)
90
Addgene inc ha vps34
( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), <t>Flag-VPS34</t> ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.
Ha Vps34, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/vps34/pmc09727793__mbc___33___ar131___s001-83-106-127?v=Addgene+inc
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Image Search Results


Primers used for cloning.

Journal: Nature

Article Title: A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria

doi: 10.1038/nature14412

Figure Lengend Snippet: Primers used for cloning.

Article Snippet: The plasmid containing human vps34 (Cat# SC118487) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Cloning, Sequencing

( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), Flag-VPS34 ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.

Journal: eLife

Article Title: Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors

doi: 10.7554/eLife.65910

Figure Lengend Snippet: ( A ) HEK293 cells were transiently transfected with HA-Rabex-5, and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ and Rabex-5. ( B, C ) HEK293 cells were transiently transfected with Flag-Rabaptin-5 ( B ), Flag-VPS34 ( C ), and/or Twinstrep-CapZβ, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, and/or VPS34. ( D ) Wavelength shift (nm) generated by the addition of Rapaptin-5 at indicated concentrations to biotinylated CapZ (200 μg/ml) immobilized on a streptavidin biosensor. Association was monitored for 5 min, followed by a 5 min dissociation step in assay buffer. Binding affinity constant was generated by fitting both the association and dissociation curves to a 1:1 binding model. ( E ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-EEA1 antibody, after which the colocalization coefficients (PCC or MCC) of EEA1 and RAB5A were quantified. ( F ) Control or CapZβ-knockout HeLa cells were immunostained with an anti-RAB5A or anti-Rabaptin-5 antibody. The colocalization coefficients (PCC or MCC) of Rabaptin-5 and RAB5A were quantified. ( G ) Control or CapZβ-knockout HeLa cells were transiently transfected with Rabex-5-GFP, and subjected to immunostaining with RAB5A antibody. The colocalization coefficients (PCC or MCC) of Rabex-5 and RAB5A were quantified. ( H ) The lysates of control or CapZβ-knockout cells were immunoprecipitated with an anti-Rabaptin-5 antibody, and the immunoprecipitates were then subjected to immunoblot analysis against Rabex-5, Rabaptin-5, and RAB5A. ( I ) Control or CapZβ-knockout HeLa cells were stably transfected with Flag-Rabex-5, and the cell lysates were then immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis against Rabex-5 and Rabaptin-5. ( J ) Control, ARP2-knockout, Rabaptin-5-knockout, or ARP2/Rabaptin-5-double knockout HeLa cells were immunostained with an anti-RAB5A antibody, and the number and size of the early endosomes in these cells were then quantified. The expression of Rapaptin-5 in control or Rapaptin-5-knockout HeLa cells was analyzed by immunoblot analysis. The BLI data (D) represent two independent experiments, and the rest of the blots, images, and graphs represent data from at least three independent experiments. The data are presented as mean ± SD. The difference between the two groups was assessed using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05. ** p<0.01, and *** p<0.001. Scale bar, 5 μm. BLI, biolayer interferometry; MCC, Manders colocalization coefficient; PCC, Pearson’s correlation coefficient. Figure 5—source data 1. Original immunoblots. Figure 5—source data 2. Data for 5D–5G, 5J.

Article Snippet: The primary antibodies were used as follows: GAPDH (Proteintech, 60004-1-Ig), CapZβ (Proteintech, 25043-1-AP), ARPC1B (Sigma-Aldrich, HPA004832), p16-Arc (Abcam, ab51243), N-WASP (Novus Biologicals, NBP1-82512), His-tag (ProteinTech, 66005-1-Ig), β-actin (ProteinTech, 60008-1-Ig), α-tublin (ProteinTech, 66031-1-Ig), VPS8 (ProteinTech, 15079-1-AP), λ-tublin (Sigma-Aldrich, T6557), HSC70 (Santa Cruz, sc-24), VPS34 (CST, 4263), CapZa1/a2 (Developmental Studies Hybridoma Bank, mAb 5B12.3), anti-pan actin (Cytoskeleton, AAN02-s), RAB5A (CST, 3547 or 46449), Calnexin (ProteinTech, 10427-2-AP), LAMP1 (CST, 9091), TGN46 (Bio-Rad, AHP500GT), EEA1 (CST, 3288), MYC-tag (ProteinTech, 67447-1-Ig), Flag-tag (Sigma-Aldrich, F3165), HA-tag (Sigma-Aldrich,11867423001), StrepMAB-Classic HRP conjugate (IBA, 2-1509-001), Rabex-5 (Santa Cruz, sc-166049), Rabaptin-5 (ProteinTech, 14350-1-AP), EGFR (Santa Cruz, sc-373746), goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, 31430), goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, 31460), and goat anti-rat IgG (H+L) secondary antibody (Invitrogen, 31470).

Techniques: Transfection, Incubation, Western Blot, Generated, Binding Assay, Control, Knock-Out, Immunostaining, Immunoprecipitation, Stable Transfection, Double Knockout, Expressing, Two Tailed Test

( A, B ) Twinstrep-CapZβ with Flag-Rabaptin-5 ( A ) or Flag-VPS34 ( B ) were transfected into HEK293 cells. The cell lysates were immunoprecipitated with an anti-Flag antibody, and the pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, or VPS34. ( C ) Coomassie staining of recombinant CapZα1-CapZβ, Rabaptin-5, and GST-RAB5 proteins. ( D ) The interaction between recombinant CapZ (CapZα1-CapZβ) and Rabaptin-5 proteins was assessed by isothermal titration calorimetry (ITC). ( E ) The interaction between RAB5A and Rabaptin-5 was assessed by ITC. ( F ) Flag-Rabaptin-5 and Twinstrep-CapZβ-expressing HEK293 cells were transfected with or without Rabex-5 siRNA. The cell lysates were incubated with Strep-Tactin Sepharose, and the pulldowns were subjected to immunoblot analysis against CapZβ and Rabaptin-5. ( G ) Flag-Rabex-5 and Twinstrep-CapZβ-expressing HEK293 cells were transfected with or without Rabaptin-5 siRNA. The cell lysates were incubated with Strep-Tactin Sepharose, and the pulldowns were subjected to immunoblot analysis against CapZβ and Rabex-5. The blots, images, and graphs represent data from two to three independent experiments. Figure 5—figure supplement 1—source data 1. Original immunoblots. Figure 5—figure supplement 1—source data 2. Data for 1D, 1E.

Journal: eLife

Article Title: Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors

doi: 10.7554/eLife.65910

Figure Lengend Snippet: ( A, B ) Twinstrep-CapZβ with Flag-Rabaptin-5 ( A ) or Flag-VPS34 ( B ) were transfected into HEK293 cells. The cell lysates were immunoprecipitated with an anti-Flag antibody, and the pulldowns were subjected to immunoblot analysis against CapZβ, Rabaptin-5, or VPS34. ( C ) Coomassie staining of recombinant CapZα1-CapZβ, Rabaptin-5, and GST-RAB5 proteins. ( D ) The interaction between recombinant CapZ (CapZα1-CapZβ) and Rabaptin-5 proteins was assessed by isothermal titration calorimetry (ITC). ( E ) The interaction between RAB5A and Rabaptin-5 was assessed by ITC. ( F ) Flag-Rabaptin-5 and Twinstrep-CapZβ-expressing HEK293 cells were transfected with or without Rabex-5 siRNA. The cell lysates were incubated with Strep-Tactin Sepharose, and the pulldowns were subjected to immunoblot analysis against CapZβ and Rabaptin-5. ( G ) Flag-Rabex-5 and Twinstrep-CapZβ-expressing HEK293 cells were transfected with or without Rabaptin-5 siRNA. The cell lysates were incubated with Strep-Tactin Sepharose, and the pulldowns were subjected to immunoblot analysis against CapZβ and Rabex-5. The blots, images, and graphs represent data from two to three independent experiments. Figure 5—figure supplement 1—source data 1. Original immunoblots. Figure 5—figure supplement 1—source data 2. Data for 1D, 1E.

Article Snippet: The primary antibodies were used as follows: GAPDH (Proteintech, 60004-1-Ig), CapZβ (Proteintech, 25043-1-AP), ARPC1B (Sigma-Aldrich, HPA004832), p16-Arc (Abcam, ab51243), N-WASP (Novus Biologicals, NBP1-82512), His-tag (ProteinTech, 66005-1-Ig), β-actin (ProteinTech, 60008-1-Ig), α-tublin (ProteinTech, 66031-1-Ig), VPS8 (ProteinTech, 15079-1-AP), λ-tublin (Sigma-Aldrich, T6557), HSC70 (Santa Cruz, sc-24), VPS34 (CST, 4263), CapZa1/a2 (Developmental Studies Hybridoma Bank, mAb 5B12.3), anti-pan actin (Cytoskeleton, AAN02-s), RAB5A (CST, 3547 or 46449), Calnexin (ProteinTech, 10427-2-AP), LAMP1 (CST, 9091), TGN46 (Bio-Rad, AHP500GT), EEA1 (CST, 3288), MYC-tag (ProteinTech, 67447-1-Ig), Flag-tag (Sigma-Aldrich, F3165), HA-tag (Sigma-Aldrich,11867423001), StrepMAB-Classic HRP conjugate (IBA, 2-1509-001), Rabex-5 (Santa Cruz, sc-166049), Rabaptin-5 (ProteinTech, 14350-1-AP), EGFR (Santa Cruz, sc-373746), goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, 31430), goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, 31460), and goat anti-rat IgG (H+L) secondary antibody (Invitrogen, 31470).

Techniques: Transfection, Immunoprecipitation, Western Blot, Staining, Recombinant, Isothermal Titration Calorimetry, Expressing, Incubation

( A ) HEK293 cells were transfected with Twinstrep-actin, and Myc-RAB5A, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against the indicated antibodies. ( B ) F-actin levels in control, CapZβ-knockout, or Twinstrep-CapZβΔC34-reconstituted HeLa cells were visualized by phalloidin staining. ( C ) Control, CapZβ-knockout, or Twinstrep-CapZβΔC34-reconstituted HeLa cells were treated with EGF-488 for the indicated times, and processed for immunofluorescence analysis. ( D, E ) HEK293 cells were transiently transfected with Flag-Rapaptin-5 ( D ), or MYC-RAB5A/HA-Rabex5 ( E ), and/or Twinstrep-CapZβ. The cells were then treated with or without CK-636 (200 μM) overnight, and the cell lysates were incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ, Rapaptin-5, RAB5A, Rabex5, β-actin, and VPS34. The blots, images, and graphs represent data from at least three independent experiments. The difference between the two groups was calculated using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05, *** p<0.001. Scale bar, 5 μm. Figure 6—figure supplement 1—source data 1. Original immunoblots. Figure 6—figure supplement 1—source data 2. Data for 1B.

Journal: eLife

Article Title: Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors

doi: 10.7554/eLife.65910

Figure Lengend Snippet: ( A ) HEK293 cells were transfected with Twinstrep-actin, and Myc-RAB5A, and the cell lysates were then incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against the indicated antibodies. ( B ) F-actin levels in control, CapZβ-knockout, or Twinstrep-CapZβΔC34-reconstituted HeLa cells were visualized by phalloidin staining. ( C ) Control, CapZβ-knockout, or Twinstrep-CapZβΔC34-reconstituted HeLa cells were treated with EGF-488 for the indicated times, and processed for immunofluorescence analysis. ( D, E ) HEK293 cells were transiently transfected with Flag-Rapaptin-5 ( D ), or MYC-RAB5A/HA-Rabex5 ( E ), and/or Twinstrep-CapZβ. The cells were then treated with or without CK-636 (200 μM) overnight, and the cell lysates were incubated with Strep-Tactin Sepharose. The pulldowns were subjected to immunoblotting analysis against CapZβ, Rapaptin-5, RAB5A, Rabex5, β-actin, and VPS34. The blots, images, and graphs represent data from at least three independent experiments. The difference between the two groups was calculated using two-tailed Student’s t-test. Differences were considered statistically significant when p<0.05, *** p<0.001. Scale bar, 5 μm. Figure 6—figure supplement 1—source data 1. Original immunoblots. Figure 6—figure supplement 1—source data 2. Data for 1B.

Article Snippet: The primary antibodies were used as follows: GAPDH (Proteintech, 60004-1-Ig), CapZβ (Proteintech, 25043-1-AP), ARPC1B (Sigma-Aldrich, HPA004832), p16-Arc (Abcam, ab51243), N-WASP (Novus Biologicals, NBP1-82512), His-tag (ProteinTech, 66005-1-Ig), β-actin (ProteinTech, 60008-1-Ig), α-tublin (ProteinTech, 66031-1-Ig), VPS8 (ProteinTech, 15079-1-AP), λ-tublin (Sigma-Aldrich, T6557), HSC70 (Santa Cruz, sc-24), VPS34 (CST, 4263), CapZa1/a2 (Developmental Studies Hybridoma Bank, mAb 5B12.3), anti-pan actin (Cytoskeleton, AAN02-s), RAB5A (CST, 3547 or 46449), Calnexin (ProteinTech, 10427-2-AP), LAMP1 (CST, 9091), TGN46 (Bio-Rad, AHP500GT), EEA1 (CST, 3288), MYC-tag (ProteinTech, 67447-1-Ig), Flag-tag (Sigma-Aldrich, F3165), HA-tag (Sigma-Aldrich,11867423001), StrepMAB-Classic HRP conjugate (IBA, 2-1509-001), Rabex-5 (Santa Cruz, sc-166049), Rabaptin-5 (ProteinTech, 14350-1-AP), EGFR (Santa Cruz, sc-373746), goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, 31430), goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, 31460), and goat anti-rat IgG (H+L) secondary antibody (Invitrogen, 31470).

Techniques: Transfection, Incubation, Western Blot, Control, Knock-Out, Staining, Immunofluorescence, Two Tailed Test

Plasmids used in this study.

Journal: eLife

Article Title: Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors

doi: 10.7554/eLife.65910

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: The primary antibodies were used as follows: GAPDH (Proteintech, 60004-1-Ig), CapZβ (Proteintech, 25043-1-AP), ARPC1B (Sigma-Aldrich, HPA004832), p16-Arc (Abcam, ab51243), N-WASP (Novus Biologicals, NBP1-82512), His-tag (ProteinTech, 66005-1-Ig), β-actin (ProteinTech, 60008-1-Ig), α-tublin (ProteinTech, 66031-1-Ig), VPS8 (ProteinTech, 15079-1-AP), λ-tublin (Sigma-Aldrich, T6557), HSC70 (Santa Cruz, sc-24), VPS34 (CST, 4263), CapZa1/a2 (Developmental Studies Hybridoma Bank, mAb 5B12.3), anti-pan actin (Cytoskeleton, AAN02-s), RAB5A (CST, 3547 or 46449), Calnexin (ProteinTech, 10427-2-AP), LAMP1 (CST, 9091), TGN46 (Bio-Rad, AHP500GT), EEA1 (CST, 3288), MYC-tag (ProteinTech, 67447-1-Ig), Flag-tag (Sigma-Aldrich, F3165), HA-tag (Sigma-Aldrich,11867423001), StrepMAB-Classic HRP conjugate (IBA, 2-1509-001), Rabex-5 (Santa Cruz, sc-166049), Rabaptin-5 (ProteinTech, 14350-1-AP), EGFR (Santa Cruz, sc-373746), goat anti-mouse IgG (H+L) secondary antibody (Invitrogen, 31430), goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, 31460), and goat anti-rat IgG (H+L) secondary antibody (Invitrogen, 31470).

Techniques: