Fig. 6

ASPA directly interacts with LYN and suppresses its phosphorylation. a Flow chart of the mass spectrometry analysis of ASPA-overexpressing PC-3 cells and control cells. b Five overlapping proteins were identified between ASPA binding proteins (306) and kinase proteins (511) in HEK293T cells. c Co-IP analysis of the interaction between ASPA and the indicated Flag-tagged five kinase proteins in PC-3 cells. d Exogenous IP assays were performed to evaluate the binding of ASPA and LYN in PC-3 cells transfected with Flag-LYN or Flag-ASPA. e Endogenous IP assays were performed in HEK293T cells transfected with Flag-LYN or HA-ASPA. f GST pull-down assays showed the direct binding of ASPA to LYN using GST-HA-ASPA and Flag-LYN (left) or using GST-HA-LYN and Flag-ASPA (right). g The colocalization of ASPA and LYN in PC-3 cells was determined by immunofluorescence (scale bar = 100 μm). h Western blotting results (left) and quantification results (right) of the phosphorylation of LYN in PC-3 cells transfected with control and ASPA overexpression vector or shRNA and shASPA. Protein expression levels were normalized to β-actin levels. i Western blotting results (left) and quantification results (right) of the phosphorylation of LYN in xenograft tumors derived from PC-3 cells transfected with control or ASPA overexpression vector or shRNA and shASPA. Protein expression levels were normalized to β-actin levels. j Western blotting results (left) and quantification results (right) of ASPA and the phosphorylation of LYN in 6 PCa samples with paired normal tissues. Protein expression levels were normalized to β-actin levels. The data are presented as the mean ± standard deviation (SD). ASPA aspartoacylase, Co-IP coimmunoprecipitation, CSNK2A1 casein kinase 2 alpha 1, DAPI 4′,6-Diamidino-2-phenylindole dihydrochloride, FYN tyrosine-protein kinase Fyn, GAK cyclin G associated kinase, GST glutathione S-transferase, HEK-293T human embryonic kidney cell 293T, IP immunoprecipitation, LYN Lck/Yes-related novel protein tyrosine kinase, PCa prostate cancer, shRNA small hairpin RNA, SRPK1 serine/arginine-rich protein-specific kinase 1. **P < 0.01