TI - Anti-phosphopeptide antibody , P-STM as a novel tool for detecting mitotic phosphoproteins : identification of lamins A and C as two major targets . AB - A polyclonal , phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 ( PAK2 ) , based on the regulatory autophosphorylation site Thr(402) of PAK2 . In this report , we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells . Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic/ acid , and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block . Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase , but not at interphase and anaphase . Interestingly , this staining pattern was almost identical to that obtained by staining with MPM2 , a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells . However , the phosphoproteins detected by the two antibodies are quite different . Two-dimensional gel electrophoresis ( 2DE ) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight ( MALDI-TOF ) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell -specific phosphoproteins recognized by P-STM . Lamins A and C immunoprecipitated from nocodazole-treated cells , but not from untreated cells showed strong reactivity to P-STM , and this reactivity lost completely after protein phosphatase 2A treatment . In summary , our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins , which are different from those recognized by MPM2 , and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM .