Anti-Cytokeratin 16/K16 antibody [EP1615Y]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

ab76416 staining Cytokeratin 16/K16 in human skin tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary antibody.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

Overlay histogram showing HepG2 cells stained with unpurified ab76416 (red line). The cells were fixed with methanol (5 min) and then permeabilized with 0.1% PBS-Triton for 20 min. The cells were then incubated in 1x PBS/10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76416, 1/50 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Triton used under the same conditions.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

ab76416 staining Cytokeratin 16/K16 in human skeletal muscle sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1 : PBS in place of primary antibody.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

ab76416 staining Cytokeratin 16/K16 in HACAT (human keratinocyte) cell lineby intracellular flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/150. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control : Rabbit monoclonal IgG (Black)

Unlabelled control : Cell without incubation with primary antibody and secondary antibody (Blue)

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

ab76416 staining Cytokeratin 16/K16 in A431 (human epidermoid carcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody. ab7291 and ab150120 were used as counterstains for primary antibody ab75748 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1 : Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2 : Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

• WB

Lab

Western blot - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

All lanes:

Western blot - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (ab76416) at 1/10000 dilution

All lanes:

HACAT (human keratinocyte) whole cell lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a> at 1/20000 dilution

Predicted band size: 51 kDa

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• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Cytokeratin 16/K16 antibody [EP1615Y] (AB76416)

Immunocytochemistry-immunofluorescence using Anti-Cytokeratin 16/K16 antibody [EP1615Y], ab76416. Publication image from Zhu, L. et al., 2022, Nat Commun, 35440136. Legend direct from paper.

Basal cytokeratins induction is independent of mutant ER genomic binding but requires low ER expression.a Heatmap representing fold change mRNA expression (E2/veh) of six basal cytokeratins and four luminal cytokeratins in ER+ breast cancer lines from six publicly available data sets (GSE89888, GSE94493, GSE108304, GSE3834, GSE38132, and GSE50693). GREB1, PGR, and TFF1 are canonical E2-regulated genes included as positive controls. b Genomic track showing ER binding intensities at KRT5/6A/6B and KRT14/16/17 loci from ER ChIP-seq data sets of MCF7 ESR1 mutant cells. GREB1 locus serve as a positive control. c Graphic view of Pearson correlation between expression of ESR1 and each basal or luminal cytokeratin in ER+ breast tumors in TCGA (n = 808) and METABRIC (n = 1505) cohorts. Color scale and size of dots represent correlation coefficient and significance, respectively. d qRT-PCR measurement of ESR1, KRT5/6A/6B/14/16/17 mRNA levels in MCF7 WT and ESR1 mutant cells with ESR1 siRNA knockdown for 7 days. mRNA fold changes were normalized to WT cells; RPLP0 levels were measured as internal control. Each bar represents mean ± SD with three biological replicates. Data shown are representative from three independent experiments. Student’s t-test (two-sided) was used to compare the gene expression between scramble and knockdown groups. e Representative images of ER, CK5, CK16, and CK17 staining in MCF7-Y537S and D538G cells. BCKs positive cells are highlighted with white arrows. Images were taken under x20 magnification. f Dot plots quantifying the ER intensities in BCKs positive (blue) and the corresponding proximal negative (red) cells from each region. Individual data points from five different regions per group from one experiment, representative of three independent experiments are shown. Paired t-test (two-sided) was applied to compare ER intensities between BCKs positive and negative cells. Source data are provided as a Source Data file for a, c, d, f.