- 701888 - Invitrogen Antibodies KLF9 antibody

Chhunchha B, Kubo E, Singh DP
Cells 2019 Sep 27;8(10)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on extracts (30 µg of lysate) of SK-OV-3 (Lane 1), HEL (Lane 2), KARPAS 299 (Lane 3), HEK-293 (Lane 4), U-87 MG (Lane 5), Jurkat (Lane 6), Mouse Liver (Lane 7), Rat Liver (Lane 8). The blots were probed with KLF9 Recombinant Rabbit Monoclonal Antibody (Product # 701888, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 27 kDa band corresponding to KLF9 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow Cytometry analysis of endogenous KLF9 was performed on SKOV-3 cells labeled with ABfinity™ Anti-KLF9 Recombinant Rabbit Monoclonal Antibody (Product# 701888, 5 ug/ 1M cells) or with rabbit isotype control at 0.5 ug/ml and detected with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, (Alexa Fluor® 488 conjugate, Product# A27034, 0.4 ug/ml, 1:2500) as represented by the red and pink histograms respectively. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control. A representative of 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer (4468770).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Enrichment of endogenous KLF9 protein at specific gene loci using Anti-KLF9 Recombinant Rabbit Monoclonal Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-KLF9 Recombinant Rabbit Monoclonal Antibody (Product # 701888, 5 µg) on sheared chromatin from 2 million K562 cells using the "MAGnify ChIP system" kit (Product # 49-2024). Normal Rabbit IgG (1 µg) was used as a negative IP control. The purified DNA was analyzed by 7500 Fast qPCR system (Product # 4351106) with optimized PCR primer pairs for the promoters of the active NOXA region used as positive control target gene, and the region of the inactive SAT2 satellite repeat, used as negative control target gene. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 SFN induced increased Nrf2 binding to ARE sites in Klf9 promoter in dose dependent fashion. ( A ) Top panel explaining the presence of four ARE sites in Klf9 promoter and ChIP-qPCR primers containing ARE3 and ARE4. ( B and C ) An in vivo DNA binding assay revealed Nrf2/ARE interaction. hLECs treated with DMSO control or different concentration of SFN (0, 12, 18 and 24 uM) for 24 h, as indicated. ChIP assay was carried out with Nrf2 and control IgG antibodies. Pulled DNA fragments were subjected to qPCR analysis. DNA fragments present in the immunoprecipitation were amplified with primers that specifically recognize a fragment of Klf9 promoter containing ARE sites as indicated. As a negative control, ChIP with control IgG was used. * p < 0.05, ** p < 0.001; SFN treated versus DMSO control. Phi and PhiPhi denotes primer pairs used for ChIP-qPCR. Note: Predicted sites ARE1 and ARE2 by in silico analysis are false positive sites (current study and [ 26 ]).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Higher doses of SFN enforced Klf9 binding to the regulatory region of its new target gene, Prdx6 . Top panel; diagrammatic illustration of in silico analysis of Prdx6 gene promoter showing five putative repressive Klf9 binding elements (RKBE; nC A/G CCCn). hLECs treated with DMSO or different concentration of SFN for 24 h, as indicated. Pulled DNA fragments with antibodies specific to Klf9 were subjected to PCR analysis for repressive Klf9 binding elements (RKBE) as described in Materials and Methods. DNA fragments present in the immunoprecipitates were amplified with primers that recognized a fragment of Prdx6 promoter containing RKBE as indicated. Control IgG served as negative control. Primers used for amplification of specific region containing Klf9 sites (* Klf9 site 1, ** Klf9 site 2, *** Klf9 site 3, **** Klf9 site 4, ***** Klf9 site 5). Primer sequences were mentioned in the Materials and Methods section.

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