vps34 antibody  (Echelon Biosciences)

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: Binding of VPS34 to TSC1 mediates TSC2 ubiquitination and degradation, and the activation of RheB A. Western blot analysis was performed to detect the endogenous levels of TSC2 in Vector, WT_118 and H868R_07 stable lines using anti-TSC2 antibodies. B. Cycloheximide chase experiment was performed to monitor TSC2 degradation. Vector and H868R_07 stable cells were incubated with cycloheximide at 50 μg/ml for indicated times. After incubation, WCL was collected and western blotting was performed to detect the levels of TSC2 using anti-TSC2 antibody. C. Stably H868R_07 cells were treated with MG132 at 5 μM for 24h or left untreated. WCL was subjected to western blot analysis to observe the levels of TSC2 protein expression. D. COS7 cells were transiently co-transfected with plasmids encoding the indicated proteins. 48h post-transfection, Flag-tagged TSC2 and Myc-tagged proteins in WCL were detected by Western blotting using anti-Flag antibody and anti-Myc antibody, respectively. E. COS7 cells were transiently co-transfected with Vector or the indicated Myc-VPS34 constructs along with Flag-TSC2 and HA-Ubiquitin (HA-Ub). 48h post-transfection, Flag-tagged TSC2 was immunoprecipitated from WCL using an anti-Flag antibody. Ubiquitinated Flag-TSC2 was detected by anti-HA antibody and immunoprecipitated Flag-TSC2 was detected by anti-Flag antibody. Myc-tagged proteins in WCL were detected using anti-Myc antibody. F. NIH3T3 cells were transiently co-transfected with Vector or the indicated Myc-VPS34 constructs plus HA-Ubiquitin plasmid. 48h post-transfection, endogenous TSC2 was immunoprecipitated from WCL and ubiquitinated TSC2 was detected by Western blot using anti-HA antibody. The immunoprecipitated TSC2 was detected by Western blot using anti-TSC2 antibody for assessing TSC2 degradation. The protein levels of Myc-VPS34 proteins in the WCL were detected by Western blot using anti-Myc antibody. G. Expression of endogenous RheB was monitored in WCL of Vector, and WT_118 and H868R_07 cells, which were treated with rapamycin (10 nM) for 2 days or left untreated, by Western blot analysis using an anti-RheB antibody. H. RheB activation assay was performed to assess the levels of active RheB-GTP in Vector, WT_118 and H868R_07 cell clones. The RheB-GTP was detected by Western blot analysis using a rabbit polyclonal anti-RheB-GTP antibody. Quantitative analysis of RheB-GTP and actin was determined from three independent experiments and expressed as mean ± SEM (*, p

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Binding Assay, Activation Assay, Western Blot, Plasmid Preparation, Incubation, Stable Transfection, Expressing, Transfection, Construct, Immunoprecipitation, Clone Assay

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: Working model depicting VPS34 regulation of mTOR/S6K1 via recruitment of PIKFYVE and TSC1 to the plasma membrane to mediate TSC2 ubiquitination and degradation and RheB activation Refer to the discussion section for the detailed description.

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Activation Assay

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: mTOR/S6K1 signaling pathway is necessary for VPS34-induced oncogenic transformation A. VPS34-H868R_07 cells were treated with rapamycin (Rapa) for 1h at the indicated concentrations or left untreated. The levels of pT389-S6K1 in WCL were detected by Western blot analysis using antibody directed against pT389-S6K1. B. Growth profiles (1% CS) of Vector cells and H868R_07 cells treated with rapamycin (10 nM) or left untreated for the indicated time. Data are representative of two or more experiments and presented in the form of mean ± STDEV (*, p

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Transformation Assay, Western Blot, Plasmid Preparation

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: VPS34 activates mTORC1/S6K1, leading to the increase in cell size (referring to Figure 1C for the clones stably expressing vector, VPS34-WT and VPS34-H868R) A. WCL obtained from indicated stable cell clones, vector, WT_118 and H868R_07 was subjected to western blotting to monitor the phosphorylated and total expression levels of S6K1. B. Stable Vector, WT_118 and H868R_07 cells were assessed for phosphorylation of 4EBP1 at T37/46 position by Western blot analysis. Insulin-treated (100 nM for 30 min) NIH3T3 cells were used as a positive control for 4EBP1 phosphorylation. The membrane was stripped and re-probed for total 4EBP1. C. Phosphorylation of Erk1/2 and Akt (S473) were detected in stable cell clones as indicated in 1% and 10% serum containing media. Membranes was stripped and re-blotted with total Erk1/2 and Akt proteins. D. The relative cell size of the indicated stable clones was analyzed using a BD FACSCalibur flow cytometer to determine the mean FSC-H of cells for the measurement of relative cell size. E. Quantitative data of flow cytometric cell size were performed from two or more independent experiments and presented in the form of bar graph (**, p

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Clone Assay, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Positive Control, Flow Cytometry

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: VPS34 binds to TSC1 but not TSC2 A. Endogenous TSC1 was co-immunoprecipitated from NIH3T3 cell WCL using anti-TSC2 antibody. B. Endogenous TSC1, but not TSC2, was co-immunoprecipitated from NIH3T3 WCL using anti-VPS34 antibody. Endogenous TSC1, TSC2 and VPS34 in WCL were detected using their corresponding antibodies. C. NIH3T3 cells were transiently transfected with pcDNA3-Myc-Vector, pcDNA3-Myc-VPS34-WT, or pcDNA3-Myc-VPS34-H868R. Anti-Myc antibody was used to immunoprecipitate Myc-tagged proteins. Endogenous TSC1 or TSC2 in the immunoprecipitates was detected by Western blot analysis using anti-TSC1 or anti-TSC2 antibody. Expression of Myc-VPS34 proteins in WCL was detected using anti-Myc antibody. D. COS-7 cells were transiently co-transfected with the indicated plasmids. VPS34 proteins were immunoprecipitated using anti-VPS34 antibody. Myc-TSC1 and Myc-VPS34 proteins in immunoprecipitates were detected using anti-Myc antibody. Myc-TSC1 expression in WCL was detected using anti-Myc antibody. E. COS7 cells were transiently co-transfected with plasmids encoding myc-VPS34-WT or Myc-VPS34-H868R plus Flag-TSC2 plasmids. Myc-tagged VPS34 proteins were immunoprecipitated used anti-Myc antibody. Immunoprecipitates were probed with anti-Flag antibody for TSC2 and then reprobed with anti-Myc antibody for VPS34 proteins.

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: VPS34 co-localizes with TSC1 and PIKFYVE, but not with TSC2 at the plasma membrane A. NIH3T3 cells were transfected with Myc-tagged VPS34 proteins. Cells were immunostained for Myc-VPS34 (red), PIKFYVE (green) and TSC1 (magenta). Bar: 20 μm. The white color indicates colocalization of VPS34, PIKFYVE, and TSC1 (Arrows in merged images). B. Experimental procedures were essentially the same as that described in (A) except cells were immunostained for TSC2 (magenta). Yellow color indicates colocalization of VPS34 and PIKFYVE (arrows in merged images). Bar: 20 μm. All images in Figure 4 were captured on Zeiss LSM-510 Meta microscope. C. NIH3T3 cells were transfected with control and PIKFYVE siRNA, and Western blot analysis was performed to show PIKFYVE knockdown. D. NIH3T3 cells were transfected with PIKFYVE siRNA for 48h or left un-transfected. Indicated WCL were subjected to immunoprecipitation using anti-VPS34 antibody or control IgG. The levels of TSC1, PIKFYVE and VPS34 were detected by Western blotting in immunoprecipitated samples.

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Transfection, Microscopy, Western Blot, Immunoprecipitation

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: VPS34-H868R induces cell cycle progression, cellular transformation, and tumor formation in mice A. Cyclin E expression levels were determined in Vector, WT_118 and H868R_07 stable clones by Western blot analysis using anti-cyclin E antibody. B. BrdU immunofluorescence staining was done on Vector, WT_118 and H868R_07 stable cell clones. Quantitative analysis of BrdU incorporation was done by counting the BrdU-positive cells in three randomly selected microscopic fields from two or more independent experiments. Data are presented as the mean percentage of BrdU-positive cells as mean ± SEM (*, p

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Transformation Assay, Mouse Assay, Expressing, Plasmid Preparation, Clone Assay, Western Blot, Immunofluorescence, Staining, Stable Transfection, BrdU Incorporation Assay

Journal: Oncotarget

Article Title: VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation

doi: 10.18632/oncotarget.10469

Figure Lengend Snippet: VPS34-H868R exhibits upregulated lipid kinase activity A. Sequence alignment between human VPS34 and p110α was performed using the NCBI blast alignment tool. B. COS-7 cells were transiently transfected with plasmids encoding the indicated proteins. Anti-Myc antibody was used to immunoprecipitate Myc-tagged proteins. Immunoprecipitates were subjected to an in vitro lipid kinase assay. Ptdins(3)p production (pmol) for each enzymatic reaction was determined by interpolation from the standard curve. Bar graph is representative of two or more experiments and presented in the form of mean ± SEM (**, p

Article Snippet: The immunoprecipitated VPS34 proteins bound on beads were used for the kinase assay.

Techniques: Activity Assay, Sequencing, Transfection, In Vitro, Kinase Assay

Journal: Nature Communications

Article Title: Class III PI3K regulates organismal glucose homeostasis by providing negative feedback on hepatic insulin signalling

doi: 10.1038/ncomms9283

Figure Lengend Snippet: UVRAG-associated class III lipid kinase is activated by insulin. ( a ) ATG14- and UVRAG-containing Vps34 complexes were immunoprecipitated from Hepa1.6 cells grown in the presence (NR) or absence of amino acids (−AA) and assayed for lipid kinase activity. Inputs for each assay were immunoblotted to determine the amounts of the Vps34 co-immunoprecipitated. PI3P signals were densitometrically measured and normalized to Vps34 protein levels. The data are presented as a fold difference of PI3P normalized to co-immunoprecipitated Vps34 levels revealed by immunoblot for each condition. Data are means±s.e.m. ( n =4, * P

Article Snippet: Vps34 antibody used for class III PI3K in vitro activity was from Echelon Biosciences (Z-R015).

Techniques: Immunoprecipitation, Activity Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy

doi: 10.1073/pnas.1217692110

Figure Lengend Snippet: Hsp70 regulates Beclin-1–Vps34 interaction and autophagosome formation. ( A ) Identification of proteins in a PS-induced Vps34 complex. Vps34 immunoprecipitates from PS-treated MCF7 cells were separated by SDS/PAGE and silver stained. Distinct bands

Article Snippet: The following antibodies were purchased from commercial sources: anti-SUMO1 (Invitrogen), anti-Vps34 (Echelon Biosciences), anti-KAP1 (Novus), anti-V5 epitope (MBL), anti-LC3B (Cell Signaling), anti-Beclin 1 (BD Biosciences).

Techniques: SDS Page, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy

doi: 10.1073/pnas.1217692110

Figure Lengend Snippet: KAP1 is the SUMO E3 ligase for Vps34. ( A ) PS induces cytoplasmic accumulation of KAP1. MCF7 cells were treated with PS for 24 h, as indicated. ( Upper ) Immunoblot analyses of KAP1 and β-actin in cytosolic extracts. ( Lower ) Immunostaining of KAP1

Article Snippet: The following antibodies were purchased from commercial sources: anti-SUMO1 (Invitrogen), anti-Vps34 (Echelon Biosciences), anti-KAP1 (Novus), anti-V5 epitope (MBL), anti-LC3B (Cell Signaling), anti-Beclin 1 (BD Biosciences).

Techniques: Immunostaining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Acetylated hsp70 and KAP1-mediated Vps34 SUMOylation is required for autophagosome creation in autophagy

doi: 10.1073/pnas.1217692110

Figure Lengend Snippet: Role of hsp70 in autophagosome formation in breast cancer cells. ( A and B ) Amino acid-starvation induces hsp70 acetylation, Vps34 SUMOylation and enhances KAP1 binding to Vps34 and acetylated hsp70. Immunoblot analyses of Vps34 ( A ) and KAP1 ( B ) immunoprecipitates

Article Snippet: The following antibodies were purchased from commercial sources: anti-SUMO1 (Invitrogen), anti-Vps34 (Echelon Biosciences), anti-KAP1 (Novus), anti-V5 epitope (MBL), anti-LC3B (Cell Signaling), anti-Beclin 1 (BD Biosciences).

Techniques: Binding Assay