Fig 1: Integratedanalysis and validation of proteome and phosphoproteome data. (A)The Venn diagram for identified total proteins (TPs), phosphoproteins (TPPs), differential expression proteins(DEPs) and differential expression phosphoproteins (DEPPs) of prepubertal and pubertal buffalo(Bubalus bubalis) testes. (B) Validation of proteome and phosphoproteome results by Western blot analysis. ODF1 and PSMA3 (pS250) were up-regulated, NUCKS1 and LMNA (pS392) were down-regulated in puberty in the cropped gels. β-actin was used as loading control. Full-length gels are presented in Supplementary Figure S3. (C) Combination figure of TMT quantitative data and Western blot analysis results. (D) Immunohistochemical analyses of NUCKS1, LMNA (pS392), and PSMA3 (pS250) in pubertal buffalo testis. NUCKS1 was mainly expressed in the nucleus of spermatogonium and primary spermatocyte, LMNA (pS392)only located in the lumen of seminiferous tubule, whereas PSMA3 (pS250)was highly expressed in the cytoplasm and nucleus of spermatogenic cells, Sertoli cells, and leydig cells of buffalo testis. Scale bars = 50 μm.