Hunting for serine 276-phosphorylated p65

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Nuclear factor kappaB (NF-kappaB) is a key regulator of inflammatory gene expression.
• Posttranslational modifications, particularly phosphorylation of the p65 subunit, modulate NF-kappaB activity.
• Phosphorylation at serine 276 (Ser276) by protein kinase A (PKA) and MSK-1 is well-documented.

Purpose of the Study:

• To evaluate the performance of commonly used commercial antibodies against phosphorylated p65 at Ser276 (P-p65 Ser276).
• To assess the specificity and reliability of these antibodies in detecting NF-kappaB signaling.
• To inform researchers about potential limitations in interpreting data generated with these reagents.

Main Methods:

• Western Blot analysis was employed to examine antibody performance.
• Analysis focused on the detection of p65 in vivo.
• Cross-reactivity of antibodies with other proteins was investigated.

Main Results:

• At least three widely used anti-P-p65 Ser276 antibodies demonstrated cross-reactivity with other proteins.
• These antibodies did not reliably detect phosphorylated p65 in vivo via Western Blot.
• The observed cross-reactivity was with proteins regulated by PKA.

Conclusions:

• Caution is advised when interpreting data obtained using the tested anti-P-p65 Ser276 antibodies.
• The findings highlight potential inaccuracies in studies relying on these antibodies for NF-kappaB phosphorylation analysis.
• Further validation of antibodies targeting posttranslational modifications is crucial for reliable research outcomes.