FMNL2 Antibody (Mouse mAb) [A6K4] Datasheet

Biological Description

Specificity	FMNL2 Antibody (Mouse mAb) [A6K4] detects endogenous levels of total FMNL2 protein.
Background	FMNL2 is a diaphanous‑related formin that functions as an effector and upstream modulator of Rho family GTPases and acts as a central regulator of actin‑dependent processes including cell morphology, motility, invasion, and angiogenesis in epithelial and tumor contexts. The protein contains N‑terminal GTPase‑binding and FH3 regulatory regions, a proline‑rich segment, and C‑terminal FH1 and FH2 actin‑assembly domains that together support autoinhibited dimers which become activated by Rho‑family inputs and then nucleate and elongate unbranched actin filaments at the cortical cytoskeleton and leading edges. FMNL2 is enriched at the cell cortex and protrusive structures where it promotes cortical actin filament dynamics, filopodia formation, and stress‑fiber organization, thereby shaping cell polarity, lamellipodial architecture, and traction forces that drive forward movement and matrix invasion. In colorectal carcinoma, FMNL2 expression is elevated in metastatic cell lines and tumor tissues and functions as a positive regulator of motility and metastasis by enhancing cell migration, invasion through extracellular matrices, and dissemination, linking its actin‑polymerizing capacity to clinically aggressive behavior. FMNL2 integrates with RhoA signaling by directly interacting with the RhoA‑specific GEF LARG and acting upstream of RhoA to promote activation of the RhoA/ROCK axis, which in turn stimulates actin assembly, stress‑fiber formation, and serum response factor–dependent transcription, and this FMNL2–RhoA–ROCK circuit is required for lysophosphatidic acid–induced RhoA activation, actin remodeling, and invasive behavior in colorectal cancer cells. FMNL2 also links cytoskeletal remodeling to pro‑metastatic transcriptional and signaling networks by targeting the NF‑κB inhibitor COMMD10 for ubiquitin‑mediated proteasomal degradation; COMMD10 reduction allows increased nuclear localization and activity of p65 NF‑κB, which supports invasion, metastasis, and poor clinical outcomes in colorectal cancer patients. In the tumor microenvironment, FMNL2 promotes angiogenesis and metastasis of colorectal cancer by using its N‑terminal GBD/FH3 region to bind epidermal growth factor‑like protein 6, facilitating EGFL6 loading into tumor‑derived exosomes and enhancing EGFL6 paracrine signaling toward endothelial cells, where EGFL6 engages the membrane receptor CKAP4 and activates the ERK/MMP pathway to drive endothelial migration and neovascularization.

Specificity

FMNL2 Antibody (Mouse mAb) [A6K4] detects endogenous levels of total FMNL2 protein.

Background

FMNL2 is a diaphanous‑related formin that functions as an effector and upstream modulator of Rho family GTPases and acts as a central regulator of actin‑dependent processes including cell morphology, motility, invasion, and angiogenesis in epithelial and tumor contexts. The protein contains N‑terminal GTPase‑binding and FH3 regulatory regions, a proline‑rich segment, and C‑terminal FH1 and FH2 actin‑assembly domains that together support autoinhibited dimers which become activated by Rho‑family inputs and then nucleate and elongate unbranched actin filaments at the cortical cytoskeleton and leading edges. FMNL2 is enriched at the cell cortex and protrusive structures where it promotes cortical actin filament dynamics, filopodia formation, and stress‑fiber organization, thereby shaping cell polarity, lamellipodial architecture, and traction forces that drive forward movement and matrix invasion. In colorectal carcinoma, FMNL2 expression is elevated in metastatic cell lines and tumor tissues and functions as a positive regulator of motility and metastasis by enhancing cell migration, invasion through extracellular matrices, and dissemination, linking its actin‑polymerizing capacity to clinically aggressive behavior. FMNL2 integrates with RhoA signaling by directly interacting with the RhoA‑specific GEF LARG and acting upstream of RhoA to promote activation of the RhoA/ROCK axis, which in turn stimulates actin assembly, stress‑fiber formation, and serum response factor–dependent transcription, and this FMNL2–RhoA–ROCK circuit is required for lysophosphatidic acid–induced RhoA activation, actin remodeling, and invasive behavior in colorectal cancer cells. FMNL2 also links cytoskeletal remodeling to pro‑metastatic transcriptional and signaling networks by targeting the NF‑κB inhibitor COMMD10 for ubiquitin‑mediated proteasomal degradation; COMMD10 reduction allows increased nuclear localization and activity of p65 NF‑κB, which supports invasion, metastasis, and poor clinical outcomes in colorectal cancer patients. In the tumor microenvironment, FMNL2 promotes angiogenesis and metastasis of colorectal cancer by using its N‑terminal GBD/FH3 region to bind epidermal growth factor‑like protein 6, facilitating EGFL6 loading into tumor‑derived exosomes and enhancing EGFL6 paracrine signaling toward endothelial cells, where EGFL6 engages the membrane receptor CKAP4 and activates the ERK/MMP pathway to drive endothelial migration and neovascularization.

Usage Information

Application

WB

Dilution

WB

1:1000

Reactivity

Human

Source

Mouse Monoclonal Antibody

MW

123 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: 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 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: 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 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/21506128/
https://pubmed.ncbi.nlm.nih.gov/36715549/

Application Data

WB

Validated by Selleck

Lane 1: 293T, Lane 2: A549, Lane 3: IMR-32