- MA5-14912 - Invitrogen Antibodies CDK9 antibody

Submitted references Epigenetic Suppression of HIV in Myeloid Cells by the BRD4-Selective Small Molecule Modulator ZL0580.

Alamer E, Zhong C, Liu Z, Niu Q, Long F, Guo L, Gelman BB, Soong L, Zhou J, Hu H
Journal of virology 2020 May 18;94(11)

A Precision Medicine Drug Discovery Pipeline Identifies Combined CDK2 and 9 Inhibition as a Novel Therapeutic Strategy in Colorectal Cancer.

Somarelli JA, Roghani RS, Moghaddam AS, Thomas BC, Rupprecht G, Ware KE, Altunel E, Mantyh JB, Kim SY, McCall SJ, Shen X, Mantyh CR, Hsu DS
Molecular cancer therapeutics 2020 Dec;19(12):2516-2527

Structure-guided drug design identifies a BRD4-selective small molecule that suppresses HIV.

Niu Q, Liu Z, Alamer E, Fan X, Chen H, Endsley J, Gelman BB, Tian B, Kim JH, Michael NL, Robb ML, Ananworanich J, Zhou J, Hu H
The Journal of clinical investigation 2019 Jul 22;129(8):3361-3373

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: J-Lat Full Length cell lysate 40 µg was loaded on 4-15% gel and transferred to 0.45 µm PVDF Membrane. CDK9 Monoclonal Antibody (Product # MA5-14912) was diluted at 1:500 in 1xTBST buffer with 5% non fat milk and incubated overnight at 4C. Goat anti Rabbit HRP-conjugated secondary antibody was diluted at 1:2000 in 1xTBST buffer with 5% non fat milk and incubated for 1 hour at 21C.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot was performed using Anti-CDK9 Monoclonal Antibody (K.513.1) (Product # MA5-14912) and a ~43 kDa band corresponding to Cyclin-dependent kinase 9 was observed across cell lines tested . Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), A-431 (Lane 3), Hep G2 (Lane 4), HEK-293 (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: J-Lat Full Length cell lysate 40 µg was loaded on 4-15% gel and transferred to 0.45 µm PVDF Membrane. CDK9 Monoclonal Antibody (Product # MA5-14912) was diluted at 1:500 in 1xTBST buffer with 5% non fat milk and incubated overnight at 4C. Goat anti Rabbit HRP-conjugated secondary antibody was diluted at 1:2000 in 1xTBST buffer with 5% non fat milk and incubated for 1 hour at 21C.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Cyclin-dependent kinase 9 was achieved by transfecting HeLa with Cyclin-dependent kinase 9 specific siRNAs (Silencer® select Product # S2835, S2834). Western Blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Cyclin-dependent kinase 9 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The Blot was probed with CDK9 Monoclonal Antibody (K.513.1) (Product # MA5-14912, 1:1000) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). Densitometric analysis of this western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Cyclin-dependent kinase 9.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot was performed using Anti-CDK9 Monoclonal Antibody (K.513.1) (Product # MA5-14912) and a ~43 kDa band corresponding to Cyclin-dependent kinase 9 was observed across cell lines tested . Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), A-431 (Lane 3), Hep G2 (Lane 4), HEK-293 (Lane 5) were electrophoresed using NuPAGE™ 10% Bis-Tris Protein Gel (Product # NP0301BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of CDK9 in HeLa cells using a CDK9 monoclonal antibody (Product # MA5-14912) (green). Actin filaments are labeled with a fluorescent red phalloidin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometric analysis of CDK9 in Jurkat cells using a CDK9 monoclonal antibody (Product # MA5-14912) (blue) compared to a nonspecific negative control antibody (red).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of CDK9 from HeLa cells using a CDK9 monoclonal antibody (Product # MA5-14912) followed by Western blot analysis using the same antibody. (Lane 1) 5% input.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 ZL0580 inhibits Tat transactivation and key factors in HIV transcription elongation. ( A and B ) Western blot measurement of Tat and NF-kappaB ( A ) and cellular proteins involved in transcription elongation ( B ) in WT J-Lat cells 24 hours after treatment. ( C ) Co-IP analysis for binding of CDK9 to Tat or BRD4 in WT J-Lat cells 24 hours after treatment. Control IgG Co-IP and input CDK9 were used as controls. Total/input CDK9 blots in panels ( B and C ) represent the same experiment. ( D ) ELL2 protein expression in WT and BRD4-KO J-Lat cells 24 hours after treatment. ( E ) ELL2 mRNA expression by qPCR in WT J-Lat cells 24 hours after treatment. ( F ) Effect of protease inhibition by MG132 on ELL2 protein levels in WT J-Lat cells. Cells were pretreated with proteasome inhibitor MG-132 for 6 hours (bottom) or not treated (top), followed by treatment with PMA or PMA+ZL0580 (10 muM) for 18 hours. ELL2 protein was measured by Western blot. ( G ) Phosphorylated RNAPII (Ser 2 CTD) in WT (top) and BRD4-KO (bottom) J-Lat cells after different treatments. Loading control GAPDH in panel ( D and G ) represents the same experiment. ( H and I ) ChIP-qPCR analysis for recruitment of Tat ( H ) or BRD4 ( I ) to HIV 5' LTR in PMA-activated or unstimulated J-Lat cells 24 hours after treatment. ChIP using control IgG was included for normalization. qPCR was conducted to quantify the precipitated HIV 5' LTR region. Data were normalized to NC. Error bars ( E ,

MA5-14912

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