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Phospho-AS160 (T642) Recombinant Rabbit Monoclonal Antibody [JE60-97]

Usd: 385

Specification

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Catalog# HA721489

Phospho-AS160 (T642) Recombinant Rabbit Monoclonal Antibody [JE60-97]

Application

  • WB

  • IF-Cell

Reactivity

  • Human

  • Mouse

  • Rat

Conjugation

  • unconjugated

Overview

Product Name

Phospho-AS160 (T642) Recombinant Rabbit Monoclonal Antibody [JE60-97]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phosphopeptide corresponding to residues surrounding Thr642 of human AS160 protein.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell

Target Molecular Weight

Predicted band size: 147 kDa

Positive Control

HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, NIH/3T3 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, PC-12 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, NIH/3T3 cells starved overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes, 293T cells starved overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes.

Conjugation

unconjugated

Clone Number

JE60-97

RRID

AB_3072605

Product Features

Form

Liquid

Concentration

1 mg/mL.(The concentration of this product may be batch-dependent)

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000

  • IF-Cell

  • 1:100

Target

Function

AS160 (Akt substrate of 160 kDa), which was originally known as TBC1 domain family member 4 (TBC1D4), is a Rab GTPase-activating protein that in humans is encoded by the TBC1D4 gene. The 160 kD protein product was first discovered in a screen for novel substrates of the serine-threonine kinase Akt2, which phosphorylates AS160 at Thr-642 and Ser-588 after insulin stimulation. Insulin stimulation of fat and muscle cells results in translocation of the glucose transporter GLUT4 to the plasma membrane, and this translocation process is dependent on phosphorylation of AS160. The role of AS160 in GLUT4 translocation is mediated by its GTPase activating domain and interactions with Rab proteins in vesicle formation, increasing GLUT4 translocation when its GTPase activity is inhibited by Akt phosphorylation. Specifically, this inhibition activates RAB2A, RAB8A, RAB10 and RAB14. AS160 also contains a calmodulin-binding domain, and this domain mediates phosphorylation-independent glucose uptake in muscle cells.

Background References

1. Sharma M et al. AKT ISOFORMS-AS160-GLUT4: The defining axis of insulin resistance. Rev Endocr Metab Disord. 2021 Dec

2. Zheng A et al. Exercise-Induced Improvement in Insulin-Stimulated Glucose Uptake by Rat Skeletal Muscle Is Absent in Male AS160-Knockout Rats, Partially Restored by Muscle Expression of Phosphomutated AS160, and Fully Restored by Muscle Expression of Wild-Type AS160. Diabetes. 2022 Feb

Subcellular Location

Cytoplasm.

UNIPROT

SwissProt: O60343 Human

SwissProt: Q8BYJ6 Mouse

Entrez Gene: 306117 Rat

Synonyms

Acrg embryonic lethality (mouse) minimal region ortholog antibody

Acrg embryonic lethality minimal region ortholog antibody

Acrg embryonic lethality mouse minimal region ortholog antibody

Akt substrate of 160 kDa antibody

AS 160 antibody

AS160 antibody

BUB2 antibody

CDC16 antibody

KIAA0603 antibody

NIDDM5 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/1,000 dilution.<br /><br />Lane 1: NIH/3T3 cell lysate<br />Lane 2: NIH/3T3 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 147 kDa<br />Observed band size: 160 kDa<br /><br />Exposure time: 25 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/1,000 dilution.

    Lane 1: NIH/3T3 cell lysate
    Lane 2: NIH/3T3 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 147 kDa
    Observed band size: 160 kDa

    Exposure time: 25 seconds;

    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 (HA721489) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/1,000 dilution.<br /><br />Lane 1: PC-12 cell lysate<br />Lane 2: PC-12 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 147 kDa<br />Observed band size: 160 kDa<br /><br />Exposure time: 25 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/1,000 dilution.

    Lane 1: PC-12 cell lysate
    Lane 2: PC-12 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 147 kDa
    Observed band size: 160 kDa

    Exposure time: 25 seconds;

    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 (HA721489) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/1,000 dilution.<br /><br />Lane 1: HEK-293 cell lysate<br />Lane 2: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate<br />Lane 3: HEK-293 cell lysate, the membrane treated with λpp for 1 hour<br />Lane 4: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, then the membrane treated with λpp for 1 hour<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 147 kDa<br />Observed band size: 160 kDa<br /><br />Exposure time: 3 minutes;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/100,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/1,000 dilution.

    Lane 1: HEK-293 cell lysate
    Lane 2: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate
    Lane 3: HEK-293 cell lysate, the membrane treated with λpp for 1 hour
    Lane 4: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, then the membrane treated with λpp for 1 hour

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 147 kDa
    Observed band size: 160 kDa

    Exposure time: 3 minutes;

    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 (HA721489) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunocytochemistry analysis of NIH/3T3 cells starved overnight then treated with or without 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes labeling Phospho-AS160 (T642) with Rabbit anti-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) 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.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    ☑ Cell treatment (CT)

    Immunocytochemistry analysis of NIH/3T3 cells starved overnight then treated with or without 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes labeling Phospho-AS160 (T642) with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-Phospho-AS160 (T642) antibody (HA721489) 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 (M1305-2, 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.

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Immunocytochemistry analysis of 293T cells starved overnight then treated with or without 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes labeling Phospho-AS160 (T642) with Rabbit anti-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-Phospho-AS160 (T642) antibody (<a href="/products/HA721489" style="font-weight: bold;text-decoration: underline;">HA721489</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) 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.<br /><br />Beta tubulin (<a href="/products/M1305-2" style="font-weight: bold;text-decoration: underline;">M1305-2</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    ☑ Cell treatment (CT)

    Immunocytochemistry analysis of 293T cells starved overnight then treated with or without 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes labeling Phospho-AS160 (T642) with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-Phospho-AS160 (T642) antibody (HA721489) 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 (M1305-2, 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.

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