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Ab79803

Manufactured by Abcam
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Ab79803 is a primary antibody for Western Blot analysis. It detects the target protein in cell and tissue lysates.

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Lab products found in correlation

Nm 005821.2 615s1c1, by Merck Group (2 mentions) Nm 005821.2 514s1c1, by Merck Group (2 mentions) Mgc human nbr2 sequence verified cdnas, by Takara Bio (2 mentions) Plvx lentiviral vector, by Takara Bio (2 mentions) Lr gateway technology, by Thermo Fisher Scientific (2 mentions) 2 deoxy d glucose and compound c, by Merck Group (2 mentions) A 769662, by LC Laboratories (2 mentions) Reduced glutathione, by Thermo Fisher Scientific (1 mentions)

4 protocols using ab79803

1

Studying AMPK Regulation via NBR2 Knockdown

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shRNAs targeting human NBR2 (NM_005821.2-615s1c1, NM_005821.2-514s1c1) were purchased from Sigma (Note that these two shRNAs target splicing isoforms #1 and #3 of NBR2, while can still achieve good knockdown efficiency when measured by real time PCR primer set designed to detect all three splicing isoforms of NBR2). siRNA targeting AMPKα were purchased from Origene (SR303721, SR303722). All three splicing isoforms of NBR2 were obtained from Thermo Fisher Scientific (MGC human NBR2 sequence-verified cDNAs, clone ID: 6452095, 4339497, 4826858) and then were subcloned into Lentiviral vector pLVX (Clontech). AMPK α, AMPK β and AMPK γ entry plasmids were obtained from Human ORFeome V5.1 library. The entry clones were subsequently recombined into gateway-compatible destination expression vectors with Flag tag through LR Gateway Technology (Invitrogen). cDNA corresponding to 1–312 a.a. of AMPK α1 was cloned into entry vector, and was subsequently recombined into gateway-compatible destination expression vectors with V5 tag through LR Gateway Technology (Invitrogen). Active human AMPK α2 protein and active human AMPK α1+AMPK β1+AMPK γ1 protein were purchase form Abcam (ab79803, ab126916). 2-Deoxy-D-glucose and compound C were purchased from Sigma (D6134, P5499). A-769662 was purchased from LC laboratories (A-1803).
Liu X., Xiao Z.D., Han L., Zhang J., Lee S.W., Wang W., Lee H., Zhuang L., Chen J., Lin H.K., Wang J., Liang H, & Gan B. (2016). lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress. Nature cell biology, 18(4), 431-442.
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2

Studying AMPK Regulation via NBR2 Knockdown

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shRNAs targeting human NBR2 (NM_005821.2-615s1c1, NM_005821.2-514s1c1) were purchased from Sigma (Note that these two shRNAs target splicing isoforms #1 and #3 of NBR2, while can still achieve good knockdown efficiency when measured by real time PCR primer set designed to detect all three splicing isoforms of NBR2). siRNA targeting AMPKα were purchased from Origene (SR303721, SR303722). All three splicing isoforms of NBR2 were obtained from Thermo Fisher Scientific (MGC human NBR2 sequence-verified cDNAs, clone ID: 6452095, 4339497, 4826858) and then were subcloned into Lentiviral vector pLVX (Clontech). AMPK α, AMPK β and AMPK γ entry plasmids were obtained from Human ORFeome V5.1 library. The entry clones were subsequently recombined into gateway-compatible destination expression vectors with Flag tag through LR Gateway Technology (Invitrogen). cDNA corresponding to 1–312 a.a. of AMPK α1 was cloned into entry vector, and was subsequently recombined into gateway-compatible destination expression vectors with V5 tag through LR Gateway Technology (Invitrogen). Active human AMPK α2 protein and active human AMPK α1+AMPK β1+AMPK γ1 protein were purchase form Abcam (ab79803, ab126916). 2-Deoxy-D-glucose and compound C were purchased from Sigma (D6134, P5499). A-769662 was purchased from LC laboratories (A-1803).
Liu X., Xiao Z.D., Han L., Zhang J., Lee S.W., Wang W., Lee H., Zhuang L., Chen J., Lin H.K., Wang J., Liang H, & Gan B. (2016). lncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress. Nature cell biology, 18(4), 431-442.
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3

In Vitro Phosphorylation of Skp2 by AMPK

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Recombinant GST-Skp2 (WT, S256A, and S256D) protein was expressed in BL21 bacteria by transformation and induced with IPTG for 18 h at room temperature. The bacteria were lysed by sonication and subjected to protein purification. GST-Skp2 (WT, S256A, and S256D) was purified by using Glutathione-S-agarose beads (Invitrogen), and then eluted with reduced glutathione (Fisher Scientific). 10-kDa-cutoff Centricon (Millipore) were used for concentrating the proteins. Recombinant active AMPKα1β1γ1 complex purified from Sf9 cells were obtained from Abcam (ab79803). HA-AMPK α1 (CA and KD) proteins were purified by transfecting the plasmid into 293T cells, and lysates were collected after 36 h and purified with HA antibody, followed by elution with HA peptide on the protein A/G beads. Purified protein concentration was determined by Bradford assay and aliquoted to Eppendorf tube. Recombinant GST-Skp2, active AMPKα1β1γ1 complex, or HA- AMPKα1 proteins were incubated for 30 min at 30 °C in 20 μl of in vitro kinase reaction buffer (2 mM DTT, 10 mM MgCl2, 25 mM Tris-HCl at pH 7.5, 0.1 mM Na3VO4 and 5 mM β-glycerophosphate, 0.5 mM ATP). The reaction was stopped after indicated time by SDS sample buffer and subjected to immunoblotting analysis.
Han F., Li C.F., Cai Z., Zhang X., Jin G., Zhang W.N., Xu C., Wang C.Y., Morrow J., Zhang S., Xu D., Wang G, & Lin H.K. (2018). The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance. Nature Communications, 9, 4728.
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4

In Vitro AMPK Phosphorylation Assay

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In vitro phosphorylation assays were performed as described previously (Greer et al., 2007 (link)) with some modifications. Briefly, 0.1 μg of purified AMPK proteins (combination of α1/β1/γ1 subunits; Abcam, 79803) were incubated with 3 μg of H2B peptide in Kinase Reaction Buffer (20 mM HEPES pH 7.5, 5 mM MgCl2, 6.25 mM β-glycerophosphate, 1.25 mM EGTA, 125 μM ATP, and 1 mM DTT) at 30°C for 15 minutes. To determine the effect of PARylation on phosphorylation, we first performed the in vitro PARylation assays as described above. After stopping the PARylation reactions by adding 10 μM PARP inhibitor PJ34, in vitro phosphorylation assays were carried out by adding 0.1 μg recombinant AMPK complex (combination of α1/β1/γ1 subunits, Abcam, ab79803), 6.25 mM β-glycerophosphate, 1.25 mM EGTA, and 125 μM ATP to the reactions with subsequent incubation for 15 minutes at 30 °C. The reactions were then stopped by the addition of one third of a reaction volume of 4x SDS-PAGE Loading Buffer (200 mM Tris•HCl pH 6.8, 8% SDS, 40% glycerol, 4% β-mercaptoethanol, 50 mM EDTA, 0.08% bromophenol blue) followed by heating to 100°C for 5 minutes.
Huang D., Camacho C.V., Setlem R., Ryu K.W., Parameswaran B., Gupta R.K, & Kraus W.L. (2020). Functional Interplay between Histone H2B ADP-ribosylation and Phosphorylation Controls Adipogenesis. Molecular cell, 79(6), 934-949.e14.
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