Anti-Hexokinase II antibody [3D3]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [3D3] (AB104836)

ab104836 at 1/200 dilution staining Hexokinase II in human esophagus cancer by Immunohistochemistry, Paraffin-embedded tissue with DAB staining.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [3D3] (AB104836)

ab104836 at 1/200 dilution staining Hexokinase II in human lung cancer by Immunohistochemistry, Paraffin-embedded tissue with DAB staining.

• IHC-P

AbReview32319****

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Hexokinase II antibody [3D3] (AB104836)

Immunohistochemical analysis of human skin tissue, staining Hexokinase II with ab104836.

Tissue was fixed with paraformaldehyde and blocked with 10% BSA for 2 hours at room temperature; antigen retrieval was by heat mediation in citrate buffer. Samples were incubated with primary antibody (1/250 in 5% NGS + 10% BSA in PBS) for 16 hours at 4°C. An HRP-conjugated goat anti-mouse polyclonal IgG (1/250) was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Ms Hadas Pahima

• Flow Cyt

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Flow Cytometry - Anti-Hexokinase II antibody [3D3] (AB104836)

ab104836 staining Hexokinase II in K562 cells by Immunofluorescence (green) and negative control (purple).

• WB

Lab

Western blot - Anti-Hexokinase II antibody [3D3] (AB104836)

Lanes 1 - 4 : Merged signal (red and green). Green - ab104836 observed at 102 kDa. Red - loading control ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) observed at 37kDa.

ab104836 was shown to react with Hexokinase II in wild-type HEK-293T cells in western blot. The band observed in HK2 CRISPR/Cas9 edited sample lane below 102 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and HK2 CRISPR/Cas9 edited HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab104836 and ab181602 (Rabbit Anti-GAPDH antibody [EPR16891]) overnight at 4°C at a 1 in 500 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Hexokinase II antibody [3D3] (ab104836) at 1/500 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

HK2 CRISPR/Cas9 edited HEK-293T cell lysate at 20 µg

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 102 kDa

Observed band size: 102 kDa

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• WB

Unknown

Western blot - Anti-Hexokinase II antibody [3D3] (AB104836)

All lanes:

Western blot - Anti-Hexokinase II antibody [3D3] (ab104836) at 1/500 dilution

Lane 1:

Jurkat cell lysate

Lane 2:

HeLa cell lysate

Lane 3:

HEK293 cell lysate

Predicted band size: 102 kDa

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• WB

CiteAb

Western blot - Anti-Hexokinase II antibody [3D3] (AB104836)

Western Blotting using Anti-Hexokinase II antibody [3D3], ab104836. Publication image from Fang, E. et al., 2020, Theranostics, 32042322. Legend direct from paper.

MZF1-uPEP/YY1/MZF1 expression is associated with NB outcome. (A) Representative images of immunohistochemical staining showing the expression patterns of MZF1-uPEP, YY1, and MZF1 in tumor cells of NB specimens (arrowheads, brown). Scale bars : 50 µm. (B) Kaplan-Meier curves indicating overall survival of 42 NB patients with high or low MZF1-uPEP immunostaining in 88 (GSE16476) NB cases with low or high expression levels of YY1 (cutoff value=1003.1). (C and D) Western blot (C) and real-time qRT-PCR (D, normalized to β-actin) assays showing the expression of YY1, MZF1, and target genes in normal dorsal root ganglia (DG), NB tissues (n=42), and NB cell lines. (E) Mining of a public microarray dataset (GSE16476) revealing the levels of YY1 in NB tissues with different status of age, death, or INSS stages. (F) The positive expression correlation of YY1 with MZF1, HK2, or PGK1 in 88 NB cases (GSE16476). (G) The mechanisms underlying MZF1-uPEP-suppressed tumor progression : as an uORF-encoded small peptide, MZF1-uPEP directly binds to YY1 to repress its transactivation, resulting in decreased transcription of MZF1 and downstream target glycolytic genes, and reduced aerobic glycolysis and tumor progression. Log-rank test for survival comparison in B. Student's t test compared the difference in D and E. Pearson's correlation coefficient analysis for gene expression in F. * P<0.05 vs. DG. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in C and D. Bars are means and whiskers (min to max) in E.

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• WB

CiteAb

Western blot - Anti-Hexokinase II antibody [3D3] (AB104836)

Western Blotting using Anti-Hexokinase II antibody [3D3], ab104836. Publication image from Fang, E. et al., 2020, Theranostics, 32042322. Legend direct from paper.

MZF1 facilitates the transcription of glycolytic genes in NB. (A) Venn diagram indicating the identification of glycolytic genes (left panel) and transcription factors (right panel) differentially expressed in 88 NB cases (GSE16476) with various status of age, death, and INSS stages, and the over-lapping analysis with potential transcription factors regulating glycolytic genes revealed by Genomatix program. The middle panel showing the potential transcription factors regulating expression of glycolytic genes. (B) Mining of a public microarray dataset (GSE16476) revealing the MZF1 levels in NB tissues with different status of age, death, or INSS stages. (C) Kaplan-Meier curve showing overall survival of 88 NB patients (GSE16476) with high or low MZF1 expression (cutoff value=226.6). (D and E) Real-time qRT-PCR (D, normalized to β-actin, n=4) and western blot (E) assays indicating the transcript and protein levels of MZF1, ALDOC, ENO1, GPI, HK2, LDHA, or PGK1 in SH-SY5Y and BE(2)-C cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), or sh-MZF1. (F) ChIP and qPCR assays showing the binding of MZF1 to promoters of HK2 and PGK1 in SH-SY5Y, SK-N-AS, BE(2)-C, and IMR-32 cells stably transfected with mock, MZF1, sh-Scb, or sh-MZF1 (n=4). (G) Dual-luciferase assay indicating the promoter activity of HK2 and PGK1 with wild-type (WT) or mutant (Mut) MZF1 binding site in SH-SY5Y and BE(2)-C cells stably transfected with mock, MZF1, sh-Scb, or sh-MZF1 (n=6). Fisher's exact test for over-lapping analysis in A. Student's t test compared the difference in B. Log-rank test for survival comparison in C. Student's t test and ANOVA compared the difference in D, F and G. * P<0.05, ** P<0.01 vs. mock or sh-Scb. δP<0.05 vs. WT. Bars are means and whiskers (min to max) in B. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in D-G.

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• WB

CiteAb

Western blot - Anti-Hexokinase II antibody [3D3] (AB104836)

Western Blotting using Anti-Hexokinase II antibody [3D3], ab104836. Publication image from Fang, E. et al., 2020, Theranostics, 32042322. Legend direct from paper.

MZF1 promotes the aerobic glycolysis of NB cells. (A) Western blot assay indicating the expression of MZF1, HK2, and PGK1 in SH-SY5Y, SK-N-AS, BE(2)-C, and IMR-32 cells stably transfected with dCas9a control (dCas9a-CTL), dCas9a-MZF1, dCas9i control (dCas9i-CTL), or dCas9i-MZF1. (B) Seahorse tracing curves (left panel) and ECAR bars (right panel) of SH-SY5Y and BE(2)-C cells stably transfected with empty vector (mock), MZF1, scramble shRNA (sh-Scb), sh-MZF1, dCas9a-CTL, dCas9a-MZF1, dCas9i-CTL, or dCas9i-MZF1, and those treated with glucose (10 mmol·L-1), oligomycin (2 µmol·L-1), or 2-deoxyglucose (2-DG, 100 mmol·L-1) at indicated (4 replicates for each point). (C and D) Glucose uptake, lactate production, and ATP levels in SH-SY5Y (C) and BE(2)-C (D) cells stably transfected with mock, MZF1, sh-Scb, sh-MZF1, dCas9a-CTL, dCas9a-MZF1, dCas9i-CTL, or dCas9i-MZF1 (n=4). Student's t test and ANOVA compared the difference in B-D. ** P<0.01 vs. mock, sh-Scb, dCas9a-CTL, or dCas9i-CTL. Data are shown as mean ± s.e.m. (error bars) and representative of three independent experiments in A-D.

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