PTPN14 Antibody [H23F7] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: ACHN, Lane 2: MDA-MB-231, Lane 3: HT-1080

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Rat

Source
Rabbit Monoclonal Antibody

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

Predicted MW
160 kDa

Positive Control	U-138 MG cells; ACHN cells; MDA-MB-231 cells
Negative Control	HT-1080 cells

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.

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.

Datasheet & SDS

Biological Description

Specificity
PTPN14 Antibody (Rabbit mAb) [H23F7] detects endogenous levels of total PTPN14 protein.

Subcellular Location
Cell membrane, Cytoplasm, Membrane

Uniprot ID
Q15678

Clone
H23F7

Synonym(s)
CATLPH; cytoskeletal-associated protein tyrosine phosphatase; MGC126803; PEZ; non-receptor type 14; Protein-tyrosine phosphatase pez; PTN14; PTP36; PTPD2; PTPN14

Background
PTPN14 (tyrosine‑protein phosphatase non‑receptor type 14; Pez/PTPD2/PTP36) is a cytoplasmic non‑receptor protein tyrosine phosphatase of the band‑4.1/FERM‑domain subfamily that integrates cell–cell contact, cytoskeletal organization, and growth control through direct regulation of the Hippo effector YAP1 and adhesion‑associated substrates. The protein contains an N‑terminal FERM domain related to 4.1/ezrin/radixin/moesin that anchors PTPN14 to the plasma membrane and cortical actin networks, followed by extended low‑complexity linker regions harboring tandem PPXY (PY) motifs that mediate binding to WW‑domain proteins, and a C‑terminal classical PTP catalytic domain that defines it as a phosphatase but is dispensable for several of its key scaffolding functions. PTPN14 associates directly with YAP1 via its two PY motifs engaging both WW domains of YAP1, with the second WW domain contributing dominantly to this interaction; this binding relocates YAP1 from the nucleus to the cytoplasm under high cell density conditions, reduces YAP1 phosphorylation at Ser127 by upstream LATS kinases only indirectly, and suppresses YAP1 transcriptional coactivator activity toward proliferative and anti‑apoptotic targets such as CTGF and AREG, thereby enforcing contact inhibition and limiting acinar outgrowth in mammary epithelial 3D culture. The interaction with YAP1 and the ability to drive its nucleus‑to‑cytoplasm translocation depend on intact PY motifs and physical complex formation but do not require PTPN14 catalytic activity, as a Cys1121Ser phosphatase‑dead mutant retains full capacity to sequester YAP1 in the cytoplasm and to rescue aberrant acini formation caused by PTPN14 loss, whereas a PY‑motif–deleted mutant fails to bind YAP1, does not correct YAP1 nuclear accumulation, and cannot restore normal acinar architecture. PTPN14 levels and thus its restraining effect on YAP1 are controlled post‑translationally by cell density through the CRL2^LRR1^ E3 ubiquitin ligase complex: at low density, LRR1‑containing CRL2 binds PTPN14, promotes its polyubiquitylation and proteasomal degradation, and maintains low PTPN14 abundance with predominantly nuclear YAP1 and high proliferative potential; at confluence, LRR1 expression decreases, PTPN14 ubiquitination and turnover decline, PTPN14 accumulates, and YAP1 becomes retained in the cytoplasm in a manner that parallels but is mechanistically distinct from canonical Hippo kinase–14‑3‑3–dependent sequestration. PTPN14 also localizes to adherens junctions and cortical actin, where its FERM domain supports association with E‑cadherin/β‑catenin complexes, and in endothelial contexts, it mediates β‑catenin dephosphorylation at adhesion junctions, stabilizing E‑cadherin–β‑catenin linkage and contributing to barrier integrity and regulation of motility, while additional interactions with VEGFR3 and roles in lymphangiogenesis connect PTPN14 to vascular and lymphatic development. Loss of PTPN14 by shRNA in non‑transformed mammary epithelial cells drives anchorage‑independent growth, disrupts polarized acini with excessive proliferation and multiacinar structures, and these oncogenic phenotypes are reversed by concurrent YAP1 knockdown, placing YAP1 as a key downstream effector of PTPN14 tumor‑suppressive activity.

Background

PTPN14 (tyrosine‑protein phosphatase non‑receptor type 14; Pez/PTPD2/PTP36) is a cytoplasmic non‑receptor protein tyrosine phosphatase of the band‑4.1/FERM‑domain subfamily that integrates cell–cell contact, cytoskeletal organization, and growth control through direct regulation of the Hippo effector YAP1 and adhesion‑associated substrates. The protein contains an N‑terminal FERM domain related to 4.1/ezrin/radixin/moesin that anchors PTPN14 to the plasma membrane and cortical actin networks, followed by extended low‑complexity linker regions harboring tandem PPXY (PY) motifs that mediate binding to WW‑domain proteins, and a C‑terminal classical PTP catalytic domain that defines it as a phosphatase but is dispensable for several of its key scaffolding functions. PTPN14 associates directly with YAP1 via its two PY motifs engaging both WW domains of YAP1, with the second WW domain contributing dominantly to this interaction; this binding relocates YAP1 from the nucleus to the cytoplasm under high cell density conditions, reduces YAP1 phosphorylation at Ser127 by upstream LATS kinases only indirectly, and suppresses YAP1 transcriptional coactivator activity toward proliferative and anti‑apoptotic targets such as CTGF and AREG, thereby enforcing contact inhibition and limiting acinar outgrowth in mammary epithelial 3D culture. The interaction with YAP1 and the ability to drive its nucleus‑to‑cytoplasm translocation depend on intact PY motifs and physical complex formation but do not require PTPN14 catalytic activity, as a Cys1121Ser phosphatase‑dead mutant retains full capacity to sequester YAP1 in the cytoplasm and to rescue aberrant acini formation caused by PTPN14 loss, whereas a PY‑motif–deleted mutant fails to bind YAP1, does not correct YAP1 nuclear accumulation, and cannot restore normal acinar architecture. PTPN14 levels and thus its restraining effect on YAP1 are controlled post‑translationally by cell density through the CRL2^LRR1^ E3 ubiquitin ligase complex: at low density, LRR1‑containing CRL2 binds PTPN14, promotes its polyubiquitylation and proteasomal degradation, and maintains low PTPN14 abundance with predominantly nuclear YAP1 and high proliferative potential; at confluence, LRR1 expression decreases, PTPN14 ubiquitination and turnover decline, PTPN14 accumulates, and YAP1 becomes retained in the cytoplasm in a manner that parallels but is mechanistically distinct from canonical Hippo kinase–14‑3‑3–dependent sequestration. PTPN14 also localizes to adherens junctions and cortical actin, where its FERM domain supports association with E‑cadherin/β‑catenin complexes, and in endothelial contexts, it mediates β‑catenin dephosphorylation at adhesion junctions, stabilizing E‑cadherin–β‑catenin linkage and contributing to barrier integrity and regulation of motility, while additional interactions with VEGFR3 and roles in lymphangiogenesis connect PTPN14 to vascular and lymphatic development. Loss of PTPN14 by shRNA in non‑transformed mammary epithelial cells drives anchorage‑independent growth, disrupts polarized acini with excessive proliferation and multiacinar structures, and these oncogenic phenotypes are reversed by concurrent YAP1 knockdown, placing YAP1 as a key downstream effector of PTPN14 tumor‑suppressive activity.

References
https://pubmed.ncbi.nlm.nih.gov/22948661/ https://pubmed.ncbi.nlm.nih.gov/20826270/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%

PTPN14 Antibody (Rabbit mAb) [H23F7]