AbstractThe search of new pharmacological targets with original mechanism of action within the ubiquitin‐proteasome pathway is still a goal to be reached in oncopharmacology. Modification by phosphorylation/dephosphorylation has been found to be involved in cancer and to regulate functional activity of proteasome. Until now, phosphorylated forms of α subunits of the 20S human proteasome have been mostly reported. Here, we have rationally designed a polyclonal antibody specifically directed against a phosphorylated peptide sequence bearing the β7 subunit Ser249 residue of the human 20S proteasome. This anti‐β7 phosphoSer249 antibody appeared to be a probe of choice to detect the presence of a phosphorylated isoform of the β7 subunit of the human 20S proteasome using mono or two‐dimensional gel electrophoresis. PhosphoSer249 was sensitive to acid phosphatase treatment of native 20S proteasome. Dephosphorylation affected the peptidylglutamyl‐peptide hydrolyzing activity whereas the chymotrypsin‐like and trypsin‐like activities remained unchanged. A comparative analysis between human normal and tumor cells showed a differential expression of the phosphoSer249 β7 isoform with a significantly lower detection in the proteasome isolated from tumor cells, suggesting its possible use as a biomarker.