38-2100

antibody from Invitrogen Antibodies

Targeting: PIK3C3 Vps34

Provider product page for 38-2100

Western blot

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [24]
Comments [0]
Validations
Immunohistochemistry [2]
Flow cytometry [1]
Other assay [27]

Submit

Product number: 38-2100 - Provider product page
Provider: Invitrogen Antibodies
Product name: VPS34 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

PIK3C3 protein structure - 38-2100 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of VPS34 showing staining in the cytoplasm of paraffin-embedded human skeletal muscle tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Anti- VPS34 Polyclonal Antibody (Product # 38-2100) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of VPS34 showing staining in the cytoplasm of paraffin-embedded mouse skeletal muscle tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Anti- VPS34 Polyclonal Antibody (Product # 38-2100) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Atg14 is recruited to Cx43-enriched plasma membrane regions during nutritional deprivation ( a ) Immunoblot for the indicated proteins in homogenates and purified plasma membrane from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2+0.2), Beclin-1 (0.7+0.5), Vps34 (0.9+0.1), Atg16 (1.1+0.3), Atg14 (2.5+0.2) and only Atg14 were significant for p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 Cx43 modulates formation of pre-autophagosomal structures through direct interaction with autophagy-related proteins ( a ) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the boxed regions are shown (bottom). Nuclei are highlighted with DAPI. ( b, c ) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum ( b ) or treated or not with 3-methyladenine ( c ) Atg5 is shown as negative control for IP. ( d ) Co-staining for Atg9 and e-cadherin in NRK cells control and knocked down for Cx43. Single and merged channels at higher magnification areas are shown. ( e ) Immunofluorescence for endogenous Atg9 and Cx43 in NRK cells control or knocked down for Eps15. ( f, g ) Immunofluorescence for endogenous Cx43 (green) in NRK cells control or knocked down for Atg14 ( f ) or Atg9 ( g ) maintained in the presence or absence of serum. ( h ) Immunofluorescence of endogenous Cx43 in NRK cells control or knocked down for Atg14 treated (or not) with Tx or Lnd (left). e-cadherin staining (red) is shown to highlight plasma membrane in g and h . Full fields are shown in Supplementary Fig. 6h . Quantification of intracytoplasmic Cx43-positives vesicles (right) (n= 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean+s.e.m and significant for (*) p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Inhibition of autophagosomal turnover leads to a reduction in Becn1 and Pik3c3 levels. A-E. B103/hAPP cells were treated with vehicle (DMSO) or 50 nM BafA for 24 hrs to inhibit autophagosomal degradation. Western blots (A) and quantification (B-E) of RIPA cell lysates that were probed with CT15 APP, LC3, Becn1, and Pik3c3 antibody. An actin antibody was used as a loading control. F. Confocal microscopy of B103/hAPP cells treated with vehicle (DMSO) or 100 nM BafA for 24 hrs. Cells were stained with CT20 APP antibody (magenta) and LC3 antibody (cyan). Co-localization is indicated in yellow. Arrowheads indicate LC3 positive APP containing vesicles. The arrow indicates an APP containing LC3 negative vesicle (scale bar represents 10 um). The line indicates cross-section. Cyan line in the cross-section represents APP intensity, magenta line represents LC3 intensity (AU). G-L. CHO/hAPP cells were treated with vehicle (DMSO), 50 nM, or 100 nM BafA (WB data not shown) for 24 hrs. Western blots (G) and quantification (H-L) of RIPA cell lysates that were probed with the CT15 APP, LC3, Becn1, and Pik3c3 antibody. An actin antibody was used as a loading control. M-N. BafA and CQ treatment cause increased APP processing which in turn leads to elevated levels of secreted APP (sAPP) in the cell supernatant (M). This is quantified in (N). O. Epifluorescence microscopy of CHO/hAPP cells treated with vehicle (DMSO) or 100 nM BafA for 12 hrs. Cells were stained with the 8E5 APP antibod

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 AD brains have less BECN1 and PIK3C3 and more LC3. A-H. Comparison of protein levels in frontal cortex (gray matter) from AD brains and age matched, non-demented, non-pathological controls. Western blots (A) and quantification (B-F) of RIPA lysates that were probed with the CT20 APP, LC3, Becn1, Pik3c3, and Atg5 antibody. An actin antibody was used as a loading control. 7 AD and 10 control cases were used. BECN1 and PIK3C3 levels were significantly reduced in AD cases (B-C). A significant linear correlation exists between BECN1 and PIK3C3 levels (R = 0.86, p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Overexpression of APP does not change Becn1 or Pik3c3 protein levels. A-B. B103 cells were stably transfected with an empty plasmid (mock) or a hAPP encoding plasmid. Western blots (A) and quantification (B) of RIPA cell lysates that were probed with the CT15 APP, a Becn1, and a Pik3c3 antibody. An actin antibody was used as a loading control. C-D. CHO cells were stably transfected with an empty plasmid (mock) or a hAPP encoding plasmid. Western blots (C) and quantification (D) of RIPA cell lysates that were probed with the CT15 APP, a Becn1, and a Pik3c3 antibody. Actin antibody was used as a loading control. Bars are mean +- SEM from triplicate cultures of at least two independent experiments. * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Reduction of Becn1 impairs degradation of autophagosomes and reduces Pik3c3 levels. A-C. CHO/hAPP cells were treated with Becn1 siRNA for 48 h. Western blots (A) of RIPA cell lysates were probed with a Becn1 and LC3 antibody. An actin antibody was used as a loading control. Quantification (B) of the Becn1 band intensity and the ratio of LC3-II to LC3-I (C). D-E. CHO/hAPP cells were treated with Becn1 and Pik3c3 siRNA for 48 h. Western blots (D) and quantification (E) of RIPA cell lysates that were probed with a Becn1 and Pik3c3 antibody. An actin antibody was used as a loading control. F-G. CHO/hAPP cells were treated with Pik3c3 siRNA for 48 h. Western blots (F) and quantification (G) of RIPA cell lysates that were probed with the CT15 APP antibody and with an actin antibody as a control for loading. Bars are mean +- SEM from triplicate cultures of at least two independent experiments. * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Human rotavirus (HRV) stimulates autophagy proteins in gnotobiotic (Gn) pig ileum. Autophagy proteins in pig ileum on post-HRV inoculation day (PID) 2. Tissue lysates were analyzed for p-mTOR (Ser 2448), total mTOR, VPS34, Beclin-1,and ATG16L1 by immunoblot. LGG, Lactobacillus rhamnosus GG.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 LGG inhibits autophagy proteins enhanced by HRV in Gn pig ileum. Autophagy proteins in pig ileum treated with LGG and HRV. Ileum epithelia were scraped on PID 2. Relative protein band intensities of ATG16L1 in pig ileum were analyzed with NIH image. Data are reported as the mean +- SD. ANOVA, * P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Detection of p-mTOR, VPS34 and lysozyme proteins by immunofluorescence in pig ileum. (A) HRV increases the expression of p-mTOR (Ser 2448) in pig ileum. Red, p-mTOR. (B) HRV increases the expression of VPS34 in Gn pig ileum. Red, VPS34. (C) HRV with/without LGG increases the expression of lysozyme in Gn pig ileum. Red, lysozyme; blue, DAPI (20x magnification, scale bar 100 mum).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 NRBF2 KO livers display reduced Atg14L-linked Vps34 activity, partially impaired autophagy and focal activation of Nrf2 pathway (a) Western blotting analysis shows accumulation of p62 in the liver of NRBF2 KO mice. Livers lysates were immunoprobed with antibodies indicated. p62 protein levels are up-regulated in the KO mice liver. (b) The quantification result shows that the p62 level increased more than three-fold in the NRBF2 KO mice liver compared with WT mice liver (Data shown as mean+-SEM, P values are indicated on the figures, unpaired Student's t test; WT, n=5 mice; KO, n=4 mice). (c) Western blotting analysis shows accumulation of high molecular weight (HMW) ubiqutin in the liver of NRBF2 KO mice. Insoluble fractions of liver lysates were immunoprobed with an anti-ubiquitin antibody, n=4 mice per genotype. (d) Atg14L-linked Vps34 kinase activity is impaired in the liver of NRBF2 KO mice. Atg14L-linked Vps34 is pulled down by an anti-Atg14L antibody and subjected to kinase assay. IP products and inputs were immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34 kinase activity versus IPed Atg14L shows the significant difference between WT and NRBF2 KO mice liver (Data shown as mean+-SEM, P values are indicated on the figure, unpaired Student's t test, n=4 mice each group). (f) Focal accumulation of p62 in the liver of NRBF2 KO mice. Liver frozen sections (15 mum in thickness) were stained with an anti-p62 antibody. Focal accum

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8 Assembly of Beclin 1-Vps34 complex in WT and NRBF2 mice, and schematic model for the NRBF2 function (a) NRBF2 is critical for the assembly of Vps34-Vps15 and Atg14L-Beclin 1 complex. Brain lysate from WT and NRBF2 KO mice were subjected to Co-IP by Vps34 antibody and Atg14L antibody. The IP products were subjected to in vitro lipid kinase assay and Western blotting analysis using the indicated antibodies. (b, c) Quantification results shows that Vps34-Vps15 and Atg14L-Beclin 1 complex interactions as well as Atg14L-linked Vps34 kinase activity are dramatically impaired in NRBF2 KO mice. (Data shown as mean+-SEM, P values are indicated on the figures, unpaired Student's t test, n=4 mice in each group). (d) Over-expression of NRBF2 enhances overall Vps34 kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP. Total Vps34 was pulled down by an anti-myc antibody and subjected to kinase assay. IP products and inputs are immunoblotted using the antibodies indicated. (e) Quantification of IPed Vps34 kinase activity, Vps15 protein normalized with IPed Vps34 shows that NRBF2-CFP, but not CFP, dMIT-CFP or dCCD-CFP significantly enhance Vps34 kinase activity and Vps34-Vps15 interaction. (Data shown as mean+-SEM, P values are indicated on the figures. IPed Vps15 / IPed Vps34, one sample t test versus hypothetical mean 1, bonferroni correction, n=3; IPed Vps34 kinase activity / IPed Vps34, multiple t test followed

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1. For figure legend, see page 2211.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5. Atg9 dynamics is impaired in CAPNS1-depleted cells. (A) ShCAPNS1 and control cells were co-transfected with GFP-Atg9 and HcRed-LC3. 24 h later, the cells were analysed using a confocal microscope. Images were acquired every 2 min over a 50 min time interval. The first (T0) and last (T90) merged pictures of the 2 h time-lapse experiments are shown. Scale bars: 20 mum. Magnifications of boxed areas are shown in the right panels. Arrows indicate ATG9 vesicles adjacent to or fused with LC3 vesicles. (B) Quantification of GFP-Atg9 and HcRed-LC3 colocalization by Pearson's coefficient determination in control and CAPNS1-depleted U2OS cells, before and after thapsigargin treatment. (C) Images sequences (T21, T23, T25) of the GFP-ATG9/HcRed-LC3 time-lapse experiment made using control and CAPNS1-depleted U2OS cells. The arrow indicates one ATG9 vesicle that meets and fuses with a LC3-positive vesicle in control cells. In shCAPNS1 cells, ATG9- and LC3-positive vesicles are more stationary. (D) U2OS cells stably expressing Flag-Atg9 were transfected with a CAPNS1-specific siRNA or a control siRNA. 48 h later, the cells were treated for 1 h with DMSO or 100 nM thapsigargin before lysis and immunoprecipitation with an anti-Flag antibody. The immunoprecipitation products were analysed by western blot to visualize Flag-Atg9, endogenous TfR and Vps34.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Interaction of BMP-2 and hVps34. HT-29 colonospheres were treated with AGG (100 ng/mL) for 72 h and the interaction of BMP-2 (Red) and hVps34 (Green) was analyzed by confocal microscopy (A). The colocalization parameters were analyzed by using JaCoP plugin in ImageJ software. HT-29 colonospheres were treated with AGG (ng/mL) for 72 h and immunoprecipitated with anti-BMP-2 and anti-hVps34 followed by immunoblotting with anti-Vps34 or anti-BMP-2 antibodies (B). A schematic ribbon representation of the BMP-2-hVps34 complex structure is shown in different colors. The hVps34 domain is shown in green color while the BMP-2 structure is shown in violet color. The residues showing the interactions are shown in sticks along the domain interface for both BMP-2 and hVps34 (C). The decomposition of DeltaG on per-residue basis or the pair interaction energy between BMP-2 and hVps34: (a) the contribution of each residue in hVps34 to BMP-2 domain binding; (b) the contribution of each residue in the BMP-2 domain to hVps34 binding (D). The distributions of the identified hotspot residues on the hVps34 (E) and BMP-2 (F) domain cartoon representation. Colored bars show the range of contributions by the residues in the unit kcal/mol

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 LPS reduces the expression of Vps34, resulting in the inhibition of omegasome formation and phagophore formation in N9 microglial cells. a The protein levels of Vps34 and Beclin1 were detected by western blotting in LPS-treated N9 microglial cells and quantified ( b , c ). d The expression of LC3 and SQSTM1 were detected by western blotting in Vps34 overexpressing N9 microglial cells after treatment with 1 mug/mL LPS for 6 h, 12 h, and 24 h, and quantified ( e , f ). g N9 microglial cells were transfected with GFP-DFCP1 and RFP-WIPI2 plasmids for 24 h and then treated with or without 1 mug/mL LPS for 12 h, and visualized by confocal microscopy. Immunofluorescence images show the colocalization of DFCP1 (green) with WIPI2 (red) in the CTRL and LPS-treated cells. The boxed areas are enlarged in the right panels. Scale bar: 10 mum. h Pearson's correlation coefficient for colocalization of DFCP1 with WIPI2 was calculated by Image J. Data are presented as mean +- SEM of three independent experiments. i Representative TEM images of N9 microglial cells cotreated with 100 nM rapamycin and 1 mug/mL LPS for 12 h. Arrows indicate phagophores (ph) and endoplasmic reticulum (ER). Scale bar: 500 nm (white), 1 mum (black). Data are presented as mean +- SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 vs control; $ p < 0.05, $$ p < 0.01 vs wild type; # p < 0.05, ## p < 0.01 vs vector