TRAF3 Antibody (Rabbit mAb) [E9G8] Datasheet

Biological Description

Specificity	TRAF3 Antibody (Rabbit mAb) [E9G8] detects endogenous levels of total TRAF3 protein.
Background	TRAF3 belongs to the TNF receptor-associated factor family of cytoplasmic adaptor proteins that couple TNF receptor superfamily members to downstream signaling cascades, and it functions as a central negative regulator of the noncanonical NF-κB pathway, a signaling branch distinct from canonical NF-κB activation in that it relies on inducible processing of the precursor protein p100 rather than degradation of IκBα. Under unstimulated conditions, newly synthesized NF-κB-inducing kinase, NIK, is immediately bound by TRAF3, which recruits it into a multi-subunit E3 ubiquitin ligase complex assembled through TRAF3 dimerization with TRAF2 and the associated ubiquitin ligases cIAP1 and cIAP2; within this complex, cIAP1/2 catalyze K48-linked ubiquitination of NIK, targeting it for continuous proteasomal degradation and keeping noncanonical NF-κB signaling switched off in resting cells. Receptor engagement by ligands such as CD40 or BAFF recruits TRAF2, TRAF3, and cIAP1/2 to the receptor complex, where TRAF2 becomes activated, likely through receptor-induced aggregation, and mediates K63-linked ubiquitination of cIAP1/2, a modification that stimulates the K48-specific ligase activity of cIAP1/2 toward TRAF3 itself rather than toward NIK. This redirected ligase activity results in K48-linked ubiquitination and proteasomal degradation of TRAF3, and because TRAF3 is required to deliver NIK to the destruction complex, its loss allows newly synthesized NIK to escape degradation and accumulate, at which point NIK phosphorylates IKKα, which in turn phosphorylates p100, triggering its ubiquitination, partial proteasomal processing to p52, and nuclear translocation of p52-RelB NF-κB dimers. TRAF2 and TRAF3 play nonredundant, complementary roles within this ubiquitination cascade, with TRAF3 serving as the direct NIK-recruiting adaptor and TRAF2 serving as the bridge that couples TRAF3 to the cIAP ligases, while cIAP1 and cIAP2 function redundantly with each other, since simultaneous inhibition of both is required to trigger noncanonical NF-κB activation. TRAF3 deficiency in mice is lethal, and this lethality can be rescued by concurrent loss of NIK, directly demonstrating that unrestrained NIK accumulation, rather than loss of TRAF3 function generally, drives the pathological consequence of TRAF3 loss. Genetic deficiencies affecting the TRAF3-TRAF2-cIAP destruction complex, or amplification of the NIK gene itself, are associated with aberrant noncanonical NF-κB activation and B-cell malignancies, particularly multiple myeloma, positioning TRAF3 as a defined checkpoint whose loss of function in B lymphocytes converts a tightly restrained kinase into a constitutively active driver of NF-κB-dependent B-cell survival and proliferation signaling.

Specificity

TRAF3 Antibody (Rabbit mAb) [E9G8] detects endogenous levels of total TRAF3 protein.

Background

TRAF3 belongs to the TNF receptor-associated factor family of cytoplasmic adaptor proteins that couple TNF receptor superfamily members to downstream signaling cascades, and it functions as a central negative regulator of the noncanonical NF-κB pathway, a signaling branch distinct from canonical NF-κB activation in that it relies on inducible processing of the precursor protein p100 rather than degradation of IκBα. Under unstimulated conditions, newly synthesized NF-κB-inducing kinase, NIK, is immediately bound by TRAF3, which recruits it into a multi-subunit E3 ubiquitin ligase complex assembled through TRAF3 dimerization with TRAF2 and the associated ubiquitin ligases cIAP1 and cIAP2; within this complex, cIAP1/2 catalyze K48-linked ubiquitination of NIK, targeting it for continuous proteasomal degradation and keeping noncanonical NF-κB signaling switched off in resting cells. Receptor engagement by ligands such as CD40 or BAFF recruits TRAF2, TRAF3, and cIAP1/2 to the receptor complex, where TRAF2 becomes activated, likely through receptor-induced aggregation, and mediates K63-linked ubiquitination of cIAP1/2, a modification that stimulates the K48-specific ligase activity of cIAP1/2 toward TRAF3 itself rather than toward NIK. This redirected ligase activity results in K48-linked ubiquitination and proteasomal degradation of TRAF3, and because TRAF3 is required to deliver NIK to the destruction complex, its loss allows newly synthesized NIK to escape degradation and accumulate, at which point NIK phosphorylates IKKα, which in turn phosphorylates p100, triggering its ubiquitination, partial proteasomal processing to p52, and nuclear translocation of p52-RelB NF-κB dimers. TRAF2 and TRAF3 play nonredundant, complementary roles within this ubiquitination cascade, with TRAF3 serving as the direct NIK-recruiting adaptor and TRAF2 serving as the bridge that couples TRAF3 to the cIAP ligases, while cIAP1 and cIAP2 function redundantly with each other, since simultaneous inhibition of both is required to trigger noncanonical NF-κB activation. TRAF3 deficiency in mice is lethal, and this lethality can be rescued by concurrent loss of NIK, directly demonstrating that unrestrained NIK accumulation, rather than loss of TRAF3 function generally, drives the pathological consequence of TRAF3 loss. Genetic deficiencies affecting the TRAF3-TRAF2-cIAP destruction complex, or amplification of the NIK gene itself, are associated with aberrant noncanonical NF-κB activation and B-cell malignancies, particularly multiple myeloma, positioning TRAF3 as a defined checkpoint whose loss of function in B lymphocytes converts a tightly restrained kinase into a constitutively active driver of NF-κB-dependent B-cell survival and proliferation signaling.

Usage Information

Application

WB

Dilution

WB

1:1000

Reactivity

Mouse, Rat, Human

Source

Rabbit Monoclonal Antibody

MW

64 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

Experimental 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes. 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) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 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) , sonicate to lyse the cells, and incubate on ice for 30 minutes. 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: 10%. 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 primary antibody dilution buffer 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.

Experimental 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.

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) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

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) , sonicate to lyse the cells, and incubate on ice for 30 minutes.

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: 10%. 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 primary antibody dilution buffer 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/22435551/
https://pubmed.ncbi.nlm.nih.gov/18997794/

Application Data

WB

Validated by Selleck

Lane 1: 293T, Lane 2: NIH/3T3, Lane 3: RAW264.7, Lane 4: Mouse brain