Datasheet

Biological Description

Specificity	UNC5B Antibody [H18P14] detects endogenous levels of total UNC5B protein.
Background	UNC5B, or Unc-5 homolog B, is a member of the UNC5 family of Netrin-1 dependence receptors with an approximate molecular weight of one hundred ten kilodaltons. It features an extracellular region containing two immunoglobulin-like domains and two thrombospondin type one repeats that facilitate binding to the Netrin-1 ligand, a single transmembrane domain, and a cytoplasmic region that includes ZU5, UPA, and death domains. These domains assemble into an inactive L-shaped closed conformation, and the cytoplasmic tail contains a caspase-3 cleavage site that enables apoptosis signaling. In the absence of Netrin-1, UNC5B induces apoptosis through ZU5, UPA, and death domain interactions that activate DAPK1 and downstream caspase cascades. When Netrin-1 binds, UNC5B recruits DCC or the protein CIP2A, which inhibits the phosphatase PP2A, blocking DAPK1 dephosphorylation, and instead activates the PI3K/Akt pathway, leading to phosphorylation of PIKE-A at serine four hundred seventy-two and MDM2 at serines one hundred sixty-six and one hundred eighty-six. UNC5B mediates repulsive axon guidance in neuronal growth cones and endothelial tip cell filopodia through cytoskeletal retraction via Rac and Rho GTPases, thereby regulating nervous system development, vascular morphogenesis, and inhibiting sprouting angiogenesis. An endothelial cell-specific splicing isoform called UNC5B delta eight, produced through NOVA2-dependent exon skipping, lacks sensitivity to Netrin-1 and constitutively drives apoptosis, which is essential for blood vessel remodeling and is associated with poor outcomes in colon cancer due to promotion of tumor angiogenesis. Dysregulation of UNC5B can promote tumor survival when Netrin-1 overexpression blocks the death pathway, as well as drive metastasis and neurodevelopmental disorders.

Specificity

UNC5B Antibody [H18P14] detects endogenous levels of total UNC5B protein.

Background

UNC5B, or Unc-5 homolog B, is a member of the UNC5 family of Netrin-1 dependence receptors with an approximate molecular weight of one hundred ten kilodaltons. It features an extracellular region containing two immunoglobulin-like domains and two thrombospondin type one repeats that facilitate binding to the Netrin-1 ligand, a single transmembrane domain, and a cytoplasmic region that includes ZU5, UPA, and death domains. These domains assemble into an inactive L-shaped closed conformation, and the cytoplasmic tail contains a caspase-3 cleavage site that enables apoptosis signaling. In the absence of Netrin-1, UNC5B induces apoptosis through ZU5, UPA, and death domain interactions that activate DAPK1 and downstream caspase cascades. When Netrin-1 binds, UNC5B recruits DCC or the protein CIP2A, which inhibits the phosphatase PP2A, blocking DAPK1 dephosphorylation, and instead activates the PI3K/Akt pathway, leading to phosphorylation of PIKE-A at serine four hundred seventy-two and MDM2 at serines one hundred sixty-six and one hundred eighty-six. UNC5B mediates repulsive axon guidance in neuronal growth cones and endothelial tip cell filopodia through cytoskeletal retraction via Rac and Rho GTPases, thereby regulating nervous system development, vascular morphogenesis, and inhibiting sprouting angiogenesis. An endothelial cell-specific splicing isoform called UNC5B delta eight, produced through NOVA2-dependent exon skipping, lacks sensitivity to Netrin-1 and constitutively drives apoptosis, which is essential for blood vessel remodeling and is associated with poor outcomes in colon cancer due to promotion of tumor angiogenesis. Dysregulation of UNC5B can promote tumor survival when Netrin-1 overexpression blocks the death pathway, as well as drive metastasis and neurodevelopmental disorders.

Usage Information

Application

WB, IP

Dilution

WB	IP
1:1000	1:50

Reactivity

Human, Mouse, Rat

Source

Rabbit Monoclonal Antibody

MW

130 kDa

Storage Buffer

PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage
(from the date of receipt)

-20°C (avoid freeze-thaw cycles), 2 years

Exprimental Methods
WB	Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Exprimental Methods

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

References

https://pubmed.ncbi.nlm.nih.gov/30084000/
https://pubmed.ncbi.nlm.nih.gov/34381052/

UNC5B Antibody [H18P14]

Biological Description

Usage Information

References

Application Data