D14e12 xp rabbit mab, by Cell Signaling Technology - Product details

1

Western Blot Analysis of NF-κB p65 in B16-F10 Cells

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Radioimmunoprecipitation assay (RIPA) buffer (R0278-500ML;
Sigma-Aldrich. St. Louis, MO) with 1 tablet protease inhibitors (4906837001;
Sigma-Aldrich. St. Louis, MO) per 10 mL RIPA buffer and phenylmethylsulfonyl
fluoride (8553; Cell Signaling Technology, Danvers, MA) at a final concentration
of 1 mM was used to extract proteins from B16-F10 cells. Briefly, cells were
disrupted in lysis buffer and protein lysates were obtained by centrifugation
for 10 minutes at 12,000 × g at 4°C. Protein
concentration was quantified with BCA protein assay (23225; Thermo Fisher
Scientific, Waltham, MA). Under reducing conditions, equivalent amounts of total
protein were fractionated using sodium dodecyl sulfate polyacrylamide-gel
electrophoresis. Membranes were probed with NF-κB p65 (D14E12)
XP^® Rabbit mAb (1:1,000 dilution, 8242S; Cell Signaling
Technology, Danvers, MA) and anti-beta actin antibody (1:1,000 dilution, ab8227;
Abeam, Cambridge, MA). Membranes were washed and incubated with secondary
antibody anti-rabbit HRP (1:10,000 dilution, sc-2313; Santa Cruz Biotechnology).
Bands were visualized with Pierce ECL Western blotting substrate (32106; Thermo
Fisher Scientific, Waltham, MA) with a ChemiDoc MP (Bio-Rad Laboratories,
Hercules, CA, USA). Knockdown of proteins was quantified with ImageJ (National
Institutes of Health, Bethesda, MD, USA).

Stansel T., Wickline S.A, & Pan H. (2020). NF-κB Inhibition Suppresses Experimental Melanoma Lung Metastasis. Journal of cancer science and clinical therapeutics, 4(3), 256-265.

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2

Western Blot Analysis of Protein Expression

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Whole cell lysates were prepared as described previously ¹⁸. 20 μg of denatured protein was fractionated on a NuPAGE Bis-Tris 4–12% gel (Life Technologies). Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for NF-kB p65 (D14E12 XP® Rabbit mAb #8242, Cell Signaling Technology, Beverly, MA) at 1:1000, Phospho-NF-kB p65^Ser536 (93H1 Rabbit mAb, #3033, Cell Signaling) at 1:1000, SOD2 (ab13534, Abcam, Cambridge, UK at 1:1000, GPX1 (#3206, Cell Signaling) at 1:1000 or Catalase (#8841, Cell Signaling) at 1:1000 and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). E-Cadherin, N-Cadherin, ZEB1, ZEB2 and β-actin (a loading control) were detected as described previously ^{20 (link)}.

Kinugasa H., Whelan K.A., Tanaka K., Natsuizaka M., Long A., Guo A., Chang S., Kagawa S., Srinivasan S., Guha M., Yamamoto K., St. Clair D.K., Avadhani N.G., Diehl J.A, & Nakagawa H. (2015). Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression. Oncogene, 34(41), 5229-5239.

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3

Western Blot Analysis of NF-κB p65 in B16-F10 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Radioimmunoprecipitation assay (RIPA) buffer (R0278-500ML;
Sigma-Aldrich. St. Louis, MO) with 1 tablet protease inhibitors (4906837001;
Sigma-Aldrich. St. Louis, MO) per 10 mL RIPA buffer and phenylmethylsulfonyl
fluoride (8553; Cell Signaling Technology, Danvers, MA) at a final concentration
of 1 mM was used to extract proteins from B16-F10 cells. Briefly, cells were
disrupted in lysis buffer and protein lysates were obtained by centrifugation
for 10 minutes at 12,000 × g at 4°C. Protein
concentration was quantified with BCA protein assay (23225; Thermo Fisher
Scientific, Waltham, MA). Under reducing conditions, equivalent amounts of total
protein were fractionated using sodium dodecyl sulfate polyacrylamide-gel
electrophoresis. Membranes were probed with NF-κB p65 (D14E12)
XP^® Rabbit mAb (1:1,000 dilution, 8242S; Cell Signaling
Technology, Danvers, MA) and anti-beta actin antibody (1:1,000 dilution, ab8227;
Abeam, Cambridge, MA). Membranes were washed and incubated with secondary
antibody anti-rabbit HRP (1:10,000 dilution, sc-2313; Santa Cruz Biotechnology).
Bands were visualized with Pierce ECL Western blotting substrate (32106; Thermo
Fisher Scientific, Waltham, MA) with a ChemiDoc MP (Bio-Rad Laboratories,
Hercules, CA, USA). Knockdown of proteins was quantified with ImageJ (National
Institutes of Health, Bethesda, MD, USA).

Stansel T., Wickline S.A, & Pan H. (2020). NF-κB Inhibition Suppresses Experimental Melanoma Lung Metastasis. Journal of cancer science and clinical therapeutics, 4(3), 256-265.

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4

Immunostaining of NF-κB p65 in Keratinocytes

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For immunostaining, rabbit monoclonal antibody NF-κB p65 (D14E12) XP® Rabbit mAb (Cell Signaling Technology, Danvers, USA) was used (1:400, incubated overnight in 4°C) with a secondary antibody, anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate, Cell Signaling Technology, Danvers, USA) used at 1:250 (incubated 2 hours at room temperature in the dark). Rabbit serum (10%) (Sigma-Aldrich, St. Louis, USA), used as a blocking buffer, eliminated all non-specific binding of the secondary antibody. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Slow Fade Diamond Antifade Mountant with DAPI, Molecular Probes, Eugene, OR, USA) for 1 hour in the dark. Images were taken using a fluorescent microscope (Leica) with x40 magnification. Cells were plated in chamber slides (Millicell EZ SLIDES, Merck Millipore, Billerica, MA, USA) in supplemented defined Keratinocyte-SFM medium (Gibco, Thermo Fisher Scientific, CA, USA). After a 24-hour incubation, cells were cultivated in serum-free keratinocyte medium without growth supplements for 16 hours. For further experiments, the cells were pretreated with 100 μM genistein for 2 hours, and then incubated with a combination of proinflammatory “cytokine mix” (Gibco, Thermo Fisher Scientific, CA, USA; Sigma-Aldrich, St. Louis, USA) for 30 minutes. The control cells were left untreated.

Smolińska E., Moskot M., Jakóbkiewicz-Banecka J., Węgrzyn G., Banecki B., Szczerkowska-Dobosz A., Purzycka-Bohdan D, & Gabig-Cimińska M. (2018). Molecular action of isoflavone genistein in the human epithelial cell line HaCaT. PLoS ONE, 13(2), e0192297.

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5

Western Blot Analysis of Protein Expression

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Whole cell lysates were prepared as described previously ¹⁸. 20 μg of denatured protein was fractionated on a NuPAGE Bis-Tris 4–12% gel (Life Technologies). Following electrotransfer, Immobilon-P membranes (Millipore) were incubated with primary antibodies for NF-kB p65 (D14E12 XP® Rabbit mAb #8242, Cell Signaling Technology, Beverly, MA) at 1:1000, Phospho-NF-kB p65^Ser536 (93H1 Rabbit mAb, #3033, Cell Signaling) at 1:1000, SOD2 (ab13534, Abcam, Cambridge, UK at 1:1000, GPX1 (#3206, Cell Signaling) at 1:1000 or Catalase (#8841, Cell Signaling) at 1:1000 and then with the appropriate HRP-conjugated secondary antibody (GE Healthcare, Piscataway, NJ). E-Cadherin, N-Cadherin, ZEB1, ZEB2 and β-actin (a loading control) were detected as described previously ^{20 (link)}.

Kinugasa H., Whelan K.A., Tanaka K., Natsuizaka M., Long A., Guo A., Chang S., Kagawa S., Srinivasan S., Guha M., Yamamoto K., St. Clair D.K., Avadhani N.G., Diehl J.A, & Nakagawa H. (2015). Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression. Oncogene, 34(41), 5229-5239.

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D14e12 xp rabbit mab

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5 protocols using d14e12 xp rabbit mab

Western Blot Analysis of NF-κB p65 in B16-F10 Cells

Western Blot Analysis of Protein Expression

Western Blot Analysis of NF-κB p65 in B16-F10 Cells

Immunostaining of NF-κB p65 in Keratinocytes

Western Blot Analysis of Protein Expression

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