VPS35 Recombinant Rabbit Monoclonal Antibody [PSH16-54]
Catalog# HA723858
VPS35 Recombinant Rabbit Monoclonal Antibody [PSH16-54]
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WB
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IF-Cell
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FC
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IHC-P
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IP
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Human
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Mouse
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Rat
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Green monkey
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HA751603
不含抗保成分
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unconjugated
Overview
Product Name
VPS35 Recombinant Rabbit Monoclonal Antibody [PSH16-54]
Antibody Type
Recombinant Rabbit monoclonal Antibody
Immunogen
Synthetic peptide within human VSP35 aa 760-795.
Species Reactivity
Human, Mouse, Rat, Green monkey
Validated Applications
WB, IF-Cell, FC, IHC-P, IP
Molecular Weight
Predicted band size: 92 kDa
Positive Control
SH-SY5Y cell lysate, A549 cell lysate, U-937 cell lysate, COS-1 cell lysate, Neuro-2a cell lysate, NIH/3T3 cell lysate, C2C12 cell lysate, C6 cell lysate, NR8383 cell lysate, PC-12 cell lysate, Mouse brain tissue lysate, Mouse colon tissue lysate, Rat brain tissue lysate, Rat colon tissue lysate, SH-SY5Y, Neuro-2a, C6, human brain tissue, human colon carcinoma tissue, human kidney tissue, mouse brain tissue, mouse kidney tissue, rat brain tissue, rat kidney tissue.
Conjugation
unconjugated
Clone Number
PSH16-54
Reactivity Data
| WB | IF-Cell | FC | IHC-P | IP | |
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| Human |
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| Mouse |
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| Rat |
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| Green Monkey |
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Product Features
Form
Liquid
Concentration
Storage Instructions
Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.
Storage Buffer
1*PBS (pH7.4), 0.1% BSA, 40% Glycerol, 0.2% Proclean 950.
Isotype
IgG
Purification Method
Protein A affinity purified.
Application Dilution
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WB
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1:5,000
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IF-Cell
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1:100
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IHC-P
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1:200-1:1,000
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FC
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1:1,000
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IP
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1-2μg/sample
Target
Function
VPS35 acts as a component of the retromer cargo-selective complex (CSC). The CSC is thought to be the core functional component of retromer or its complex variants. It plays a role in preventing the missorting of selected transmembrane cargo proteins into the lysosomal degradation pathway. VPS35 is involved in the recruitment of the CSC to the endosomal membrane, a process that involves RAB7A and SNX3. It is believed that the CSC associates with the cytoplasmic domain of cargo proteins mainly via VPS35, though these interactions are of low affinity. Retromer SNX proteins may also contribute to cargo selectivity. VPS35 is implicated in various retromer - mediated transport processes. The SNX - BAR retromer, which VPS35 is associated with, is involved in retrograde transport of cargo proteins from endosomes to the trans - Golgi network (TGN) and endosome - to - plasma membrane transport for cargo protein recycling. The SNX3 - retromer, also linked to VPS35, mediates the retrograde transport of WLS through a distinct pathway from the SNX - BAR retromer. The SNX27 - retromer, in which VPS35 is involved, is thought to be responsible for endosome - to - plasma membrane trafficking and recycling of a wide range of cargo proteins. VPS35 seems to function as a recruitment hub for other proteins such as the WASH complex and TBC1D5. It is required for the retrograde transport of lysosomal enzyme receptors IGF2R and SLC11A2, and plays a part in regulating the transcytosis of the polymeric immunoglobulin receptor (pIgR - pIgA). Moreover, VPS35 is necessary for the endosomal localization of WASHC2 and facilitates the association of the CSC with the WASH complex.
Background References
1. Yu J et al. VPS35 promotes cell proliferation via EGFR recycling and enhances EGFR inhibitors response in gastric cancer. EBioMedicine. 2023 Mar
2. Zhou Q et al. VPS35 promotes gastric cancer progression through integrin/FAK/SRC signalling-mediated IL-6/STAT3 pathway activation in a YAP-dependent manner. Oncogene. 2024 Jan
Subcellular Location
Cytoplasm, Membrane, Endosome, Early endosome, Late endosome.
UNIPROT
Synonyms
DKFZp434E1211 antibody
DKFZp434P1672 antibody
FLJ10752 antibody
FLJ13588 antibody
FLJ20388 antibody
hVPS35 antibody
Maternal embryonic 3 antibody
Maternal-embryonic 3 antibody
MEM 3 antibody
MEM3 antibody
ExpandImages
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Western blot analysis of VPS35 on different lysates with Rabbit anti-VPS35 antibody (HA723858) at 1/5,000 dilution.
Lane 1: SH-SY5Y cell lysate (20 µg/Lane)
Lane 2: A549 cell lysate (20 µg/Lane)
Lane 3: U-937 cell lysate (20 µg/Lane)
Lane 4: COS-1 cell lysate (20 µg/Lane)
Lane 5: Neuro-2a cell lysate (20 µg/Lane)
Lane 6: NIH/3T3 cell lysate (20 µg/Lane)
Lane 7: C2C12 cell lysate (20 µg/Lane)
Lane 8: C6 cell lysate (20 µg/Lane)
Lane 9: NR8383 cell lysate (20 µg/Lane)
Lane 10: PC-12 cell lysate (20 µg/Lane)
Lane 11: Mouse brain tissue lysate (40 µg/Lane)
Lane 12: Mouse colon tissue lysate (40 µg/Lane)
Lane 13: Rat brain tissue lysate (40 µg/Lane)
Lane 14: Rat colon tissue lysate (40 µg/Lane)
Predicted band size: 92 kDa
Observed band size: 80 kDa
Exposure time: 59 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723858) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
☑ Knockdown (KD)
Western blot analysis of VPS35 on different lysates with Rabbit anti-VPS35 antibody (HA723858) at 1/5,000 dilution.
Lane 1: HAP1-parental cell lysate (10 µg/Lane)
Lane 2: HAP1-VPS35 KD cell lysate (10 µg/Lane)
Predicted band size: 92 kDa
Observed band size: 80 kDa
Exposure time: 3 minutes; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723858) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature. -
Immunocytochemistry analysis of SH-SY5Y cells labeling VPS35 with Rabbit anti-VPS35 antibody (HA723858) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-VPS35 antibody (HA723858) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunocytochemistry analysis of Neuro-2a cells labeling VPS35 with Rabbit anti-VPS35 antibody (HA723858) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-VPS35 antibody (HA723858) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunocytochemistry analysis of C6 cells labeling VPS35 with Rabbit anti-VPS35 antibody (HA723858) at 1/100 dilution.
Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-VPS35 antibody (HA723858) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.
Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution. -
Immunohistochemical analysis of paraffin-embedded human brain tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded human colon carcinoma tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-VPS35 antibody (HA723858) at 1/200 dilution.
The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723858) at 1/200 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX. -
Flow cytometric analysis of SH-SY5Y cells labeling VPS35.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA723858, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black). -
Flow cytometric analysis of Neuro-2a cells labeling VPS35.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA723858, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black). -
Flow cytometric analysis of C6 cells labeling VPS35.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA723858, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black). -
VPS35 was immunoprecipitated from 0.2 mg A549 cell lysate with HA723858 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723858 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: A549 cell lysate (input)
Lane 2: HA723858 IP in A549 cell lysate
Lane 3: Rabbit IgG instead of HA723858 in A549 cell lysate
Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 3 minutes; ECL: K1801 -
VPS35 was immunoprecipitated from 0.2 mg NIH/3T3 cell lysate with HA723858 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723858 at 1/5,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.
Lane 1: NIH/3T3 cell lysate (input)
Lane 2: HA723858 IP in NIH/3T3 cell lysate
Lane 3: Rabbit IgG instead of HA723858 in NIH/3T3 cell lysate
Blocking/Dilution buffer: primary antibody dilution (K1803)
Exposure time: 46 seconds; ECL: K1801
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