Antibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [3]
- Other assay [7]
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Validation data
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- Product number
- PA5-17831 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-AMPK alpha-1,2 (Thr183, Thr172) Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- It is not recommended to aliquot this antibody.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 83 µg/mL
- Storage
- -20°C
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Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with Metformin (10mM for 24 h) (Lane 2), Hep G2 (Lane 3), Hep G2 treated with Metformin (10mM for 24 h) (Lane 4), U-87 MG (Lane 5), and U-87 MG treated with AICAR (1mM for 48 h) (Lane 6). The blot was probed with Phospho-AMPK alpha-1,2 (Thr183, Thr172) Monoclonal Antibody (Product # PA5-17831, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 63 kDa band corresponding to Phospho-AMPK alpha-1 (Thr172) was observed across the cell lines tested and enhanced upon treatment.
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- Western blot analysis of Phospho-AMPK-alpha 1,2 (Thr183, Thr172) was performed by loading 30 µg of THP-1 cell lysates from cells either treated with a vehicle control (C, left lane) or with 100 nM PMA (right lane) for 15 minutes, onto an SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature. The membrane was probed with a Phospho-AMPK alpha-1,2 (Thr183, Thr172) polyclonal antibody (Product # PA5-17831) at a dilution of 1:1000 for overnight at 4°C, washed in TBST, and probed with an HRP-conjugated goat anti-rabbit IgG at a dilution of 1:40,000 for 1 hour at room temperature. Detection was performed using ECL substrate. Data courtesy of the Innovators Program.
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- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with Metformin (10mM for 24 h) (Lane 2), Hep G2 (Lane 3), Hep G2 treated with Metformin (10mM for 24 h) (Lane 4), U-87 MG (Lane 5), and U-87 MG treated with AICAR (1mM for 48 h) (Lane 6). The blot was probed with Phospho-AMPK alpha-1,2 (Thr183, Thr172) Monoclonal Antibody (Product # PA5-17831, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 63 kDa band corresponding to Phospho-AMPK alpha-1 (Thr172) was observed across the cell lines tested and enhanced upon treatment.
Supportive validation
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- Figure 4 Effect of vaspin on mitogen-activated kinase (MAP3/1) and AMP-activated kinase (PRKAA1) phosphorylation in oocytes and cumulus cells after in vitro maturation. COCs (50/group/experiment) were selected after morphological examination of material collected from the ovaries taken from 100 pigs (15 follicles/ovary). Porcine cumulus-oocyte complex (COCs) were cultured for 44 h in maturation medium in the presence or absence of vaspin (1 ng/mL) then mechanically separated into oocytes and cumulus cells. Phospho MAP3/1 (pMAP3/1), MAP3/1 ( A ) and phospho PRKAA1 (pPRKAA1), PRKAA1 ( B ) protein expressions were analysed by Western blot analysis. Protein expression levels were normalised to actin. Experiments were performed independently and repeated three times ( n = 3). Data are plotted as the mean +- SEM of three independent experiments. Significance between control and vaspin is indicated by ** p < 0.01 and *** p < 0.001.
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- Fig. 3 SIRT6 enhances OXPHOS and energy status in MDA-MB-231 cells. a - h Expression ( a ) and activity ( b ) of pyruvate dehydrogenase (PDH), activity of mitochondrial complexes ( c - e ), oxygen consumption ( f ), activity of F o -F 1 ATP synthase ( g ), and energy status, expressed as ATP/AMP ratio ( h ), were measured in MDA-MB-231 cells transduced with human WT or catalytically inactive (H133Y) SIRT6, or with a control vector (VECTOR). Data are presented as mean +- SD of three different experiments. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not statistically significant. i - p Expression ( i ) and activity ( j ) of PDH, activity of mitochondrial complexes ( k - m ), oxygen consumption ( n ), activity of F o -F 1 ATP synthase ( o ), and energy status, expressed as ATP/AMP ratio ( p ) were measured in MDA-MB-231 cells transduced with a short hairpin RNA targeting SIRT6 or with a control vector (VECTOR). Data are presented as mean +- SD of three different experiments. ** P < 0.01, *** P < 0.001. q 2 x 10 6 MDA-MB-231 BC cells transduced with either a SIRT6-shRNA or a control vector (VECTOR) were injected subcutaneously in both flanks of BALB/c athymic nude mice. Mice were sacrificed, and tumors were excised 50 days after cell inoculation. Proteins were extracted, and phosphorylated AMPK (Thr183, Thr172), total AMPK, and vinculin were detected by Western blot. One representative experiment out of two is presented
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- Additional file 5: Fig. S5. SIRT6 silencing enhances AMPK phosphorylation in MDA-MB-231 xenografts. A, MDA-MB-231 cells were engineered to express a SIRT6-shRNA (or a control vector). Thereafter, cells were used for protein lysate generation and phosphorylated AMPK (Thr183, Thr172), total AMPK and GAPDH were detected by Western blot. One representative experiment out of three is presented. B, MDA-MB-231 BC cells transduced with either a SIRT6-shRNA or with a control vector were injected subcutaneously into both flanks of BALB/c athymic nude mice. Animals were sacrificed 50 days after cell inoculation; tumors were used for protein lysate generation and phosphorylated and total AMPK, SIRT6 and vinculin were detected by Western blot. In the right panel, the intensity of the phospho-AMPK bands was normalized to that of the total AMPK bands and the phospho-AMPK/total-AMPK ratio in tumors with silenced SIRT6 was compared to that detected in control tumors. Data are presented as mean +- SD. * p
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- Figure 5 Gomisin N prevents ethanol-mediated reduction in sirtuin1 (SIRT1) and phosphorylated AMP-kinase (AMPK) in the liver of chronic-binge ethanol-fed mice. ( A ) Western blot analysis of SIRT1. Representative images are shown ( left ). SIRT1 level was quantified by densitometry ( right ). ( B ) Western blot analysis of p-AMPK and acetyl-CoA carboxylase (p-ACC). Representative images are shown ( left ). Levels of p-AMPK and p-ACC were quantified by densitometry ( right ). Values are the mean +- SD ( n = 6). # p < 0.01, ## p < 0.01 vs. pair-fed control mice, * p < 0.05, ** p < 0.01 vs. ethanol-fed mice.
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- Figure 9 Gomisin N reverses ethanol-mediated reduction in SIRT1 and phosphorylated AMPK in HepG2 cells. HepG2 cells were treated with 50 mM ethanol in the presence or absence of GN (50 or 100 muM) for 24 h. ( A ) Western blot analysis of SIRT1. ( B ) Western blot analysis of p-AMPK and p-ACC. ( C ) Measurement of NAD + /NADH ratio. ( D ) HepG2 cells were pretreated with GN (100 muM) for 3 h or with Ex52735 (10 muM) for 6 h and then treated with ethanol (100 muM). Measurement of intracellular TG levels. Values are the mean +- SD of triplicate experiments. # p < 0.05, ## p < 0.01 vs. untreated control, * p < 0.05, ** p < 0.01 vs. ethanol-treated group. SS p < 0.05 vs. ethanol and GN-treated group.
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- Figure 7. Effects of CNP and cANP 4-23 , an agonist of NPR-C, on the regulation of atrial p-AMPK expression. Data were expressed as mean +- standard error of the mean; n=5. *P
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- Figure 5 CT activated AMPK/SIRT1 signaling. ( A , B ) HepG2 cells were treated with 2.5 muM CT for indicated times. Western blot analysis of phosphorylated AMPK , ACC , SIRT1 , and Nrf2 . ( C ) C57BL/6 mice were pair-fed either control or ethanol-containing diet with or without CT (20 or 40 mg/kg) for four weeks. Western blot analysis of phosphorylated AMPK , SIRT1. CYP2E1 , and Nrf2 . ( D ) HepG2 cells were incubated with 50 mM ethanol and treated with CT (2.5 or 5 muM) for 24 h. Western blot analysis of phosphorylated AMPK , SIRT1 , CYP2E1 , and Nrf2 . ( E ) AML-12 cells were incubated with 50 mM ethanol and treated with CT (2.5 or 5 muM) for 24 h. Western blot analysis of phosphorylated AMPK and SIRT1 . The images are representative ( F ) HepG2 cells were pretreated with CT (2.5 muM) for 3 h or with compound C (comp C) (10 muM) for 6 h, followed by ethanol (100 muM) treatment. Measurement of intracellular TG levels. Data are shown as mean +- SD of three independent experiments. # p < 0.05 vs. untreated control, ** p < 0.01 vs. ethanol-treated group. SS SS p < 0.01 vs. ethanol and CT-treated group. Densitometric analysis of western blots are given in Supplementary Figures S2 and S3A-G .