AtaGenix Customized Phosphorylated Polyclonal Antibodies Unveil the Role of SLKv in Cancer Glycolysis

Research Background

Abnormal energy metabolism is a hallmark of tumor cells, with the glycolysis pathway playing a particularly critical role. This study focused on an alternative splicing event of Ste20-like kinase (SLK), revealing that its variant SLKv enhances phosphoenolpyruvate (PEP) production by phosphorylating Enolase 1 (ENO1) at the Ser2 site, thereby accelerating glycolysis and driving tumor progression. Using liver cancer patient samples, cell lines, and animal models, the research team systematically elucidated the mechanism of the “SLKv–ENO1–PEP” metabolic acceleration axis. The findings were published in Cancer Research (2025, DOI: 10.1158/0008-5472.CAN-25-0523).

Client Needs

The research team required: (1) a highly specific phosphorylation antibody targeting the ENO1 Ser2 site that could distinguish phosphorylated ENO1 from the unmodified form across WB, IP, and IHC platforms; (2) recombinant ENO1 proteins — wild-type and phospho-mimetic/dead mutants (S2D/S2A) — as functional controls for mechanistic validation; and (3) supporting capabilities including phage display screening and multi-platform functional validation to confirm the antibody’s specificity and the biological significance of Ser2 phosphorylation.

Technical Challenges

Developing a phospho-specific antibody against ENO1 Ser2 presented multiple difficulties:

• Phosphorylation sites are inherently low-immunogenicity epitopes — the phospho-serine modification must be the dominant epitope recognized, not the surrounding backbone sequence.
• The antibody had to be compatible with multiple detection platforms (WB, IP, IHC) under different fixation and denaturation conditions while maintaining low background.
• Cross-reactivity with unmodified ENO1 had to be eliminated through dephospho-peptide depletion during purification.
• Recombinant ENO1 proteins (WT, S2A phospho-dead, S2D phospho-mimetic) were needed as controls, requiring stable expression with preserved enzymatic activity.

Customized Solutions

AtaGenix designed an integrated workflow combining phospho-antibody development and recombinant protein production:

• Phospho-Peptide Immunogen Design: Phosphorylated peptides were designed based on the Ser2 neighborhood sequence of ENO1 and conjugated to KLH for immunization. Both phospho and dephospho peptides were synthesized — the phospho form for immunization (KLH conjugate) and the dephospho form for negative depletion during purification.
• Two-Round Affinity Purification: Rabbit polyclonal antibodies were produced and purified through two sequential steps: positive selection on the phospho-peptide column, followed by negative depletion against the unmodified peptide column. This ensured only phospho-Ser2-specific antibodies were retained, eliminating cross-reactivity with unmodified ENO1.
• Recombinant Protein Panel: ENO1 wild-type, S2A (phospho-dead mutant), and S2D (phospho-mimetic mutant) were expressed in E. coli with multi-step purification (Ni-NTA, ion exchange, gel filtration) to provide the functional controls needed for mechanistic validation.
• Multi-Platform Validation: The phospho-ENO1(S2) antibody was validated across WB, IP, ELISA, IF, and IHC with recommended usage conditions (dilution ratios, antigen retrieval methods, blocking conditions) provided in the QC report to ensure cross-platform reproducibility.

Research Outcomes and Impact

The custom phospho-ENO1(S2) antibody enabled the research team to accurately confirm the phosphorylation status of ENO1 at key nodes of the glycolysis pathway. Combined with the S2A/S2D mutant proteins, the team obtained reproducible molecular evidence that SLKv-mediated Ser2 phosphorylation enhances ENO1 catalytic activity, increases PEP production, and accelerates glycolysis in hepatocellular carcinoma. The study further demonstrated that targeting SLKv with antisense oligonucleotides effectively inhibits glycolysis and tumor growth, establishing the SLKv–ENO1–PEP axis as a promising metabolic target for cancer therapy.

This case study is based on a published research collaboration. Results may vary depending on target antigen, modification type, and experimental conditions. All proprietary client information is subject to NDA. Reference: Cancer Research. 2025. DOI: 10.1158/0008-5472.CAN-25-0523