in addition, 35 proteins were detected by both the powerblot and kinexus systems ( table 1 ). table 1proteins detected by both powerblot and kinexus systemsprotein nameswiss nrprotein nameswiss nrbmxp51813mek2p36506cam kinase kinaseq64572mkp2q62767casein kinase i epsilonp49674p38 alpha / sapk2aq16539casein kinase ii alpha / ck2ap19139paxillinp49024cdk1 / cdc2p06493pka cp17612cdk5q00535pkc betap05771cdk7p50613pkc deltaq05655dap kinasep53355pp2a catalytic alphap05323dap3p51398pp5 / pptp53042erk1q63538ptp1bp18031erk2p27703ptp1c / shp1p29350fakq00944ptp1d / shp2q06124gsk - 3 betap18266rbp13405i kappa b alphap25963rskq15418ikk betao14920stat1a46159jak1p23458stat3p52631jnk1p45983vhrp51452mek1q02750 detection of protein isoforms or post - translational variantsunlike many cdna - based gene expression assays, western blotting has the capacity to detect multiple protein isoforms due to translation of different mrna splice variants, as well as post - translational modifications such as enzymatic cleavage, glyco sylation, or phosphorylation. examination of the blots described here identified 42 examples of multiple banding for a single target antigen ( fig. 3c ). these candidates exhibited closely migrating multiple bands, which were close to their predicted size but were sufficiently separated from other proteins. for example, four closely migrating bands were observed for fgf2 ( fig. 3c, top panel ), which may represent known glyco sylation variants of this growth factor [ 33 ]. other known examples of post - translational modifications included those of hsp70, ikkgamma and erk1. verification of protein expression by immunocytochemistrythe powerblot and kinexus assays identified proteins based on their expected and observed molecular weight, using combinations of antibod ies that had been predetermine d to detect proteins of sufficiently different sizes. proteins known to be expressed by hescs and also identified by these assays, included oct4, e - cad, connexin 43 and hsp70. to verify expression using a complementary approach, we performed immunoflu rorescent staining for 10 proteins not previously reported to be expressed in hescs by immunocytochemistry, using karyotypically normal bg01 cultures ( fig. 4a – k ). these included abp - 280, a homodimeric actin - binding protein often associated with membrane glyco proteins; ctbp1 and ctbp2, two c terminal binding proteins that are a class of transcription corepressors; gs - 28, a golgi protein; hdj - 2, a member of the dnaj - related hsp40 ( heat shock protein 40 ) subfamily; l - caldesmon, a cytoplasmic actin - binding protein; rabaptin, a gtp - binding protein; phosphorylated - p130 cas, a docking protein with an amino - terminal sh3 domain that may function as a molecular switch that regulates cas ( crk - associated substrate ) tyrosine phosphorylation; ras - gap and phosphorylated ras - gap ( p - y460 ), a protein that down-regulate s the signal transducer p21ras; and shcc, a protein with an n - terminal phosphotyrosine - binding domain. these proteins were all expressed by hescs, with the expected subcellular localization ( fig. 4a – k ). oct4 was used as a positive control ( fig. 4l ). these results suggested that most of the bands in the powerblot and kinexus assays were likely to be correctly identified. figure 4verification of protein expression using immunocytochemistry. ( a - k ) ten proteins that were detected in undifferentiated hescs by western blotting were also detected by immunoflu orescence of bg01 cells grown in mef - cm. ras - gap ( py460 ) is a phosphorylated form of ras - gap. the same antibod ies were used in this analysis as in the powerblot assay, except phospho - p130 cas ( tyr165 ). ( l ) oct4 was used as a positive control. ( m - r ) oct4, tnik and p130 cas as markers of undifferentiated hescs. bg01 cultures were partially differentiated by exposure to 10 % fetal bovine serum for 3 days. ( m ) oct4 was expressed uniformly in undifferentiated cells, ( p ) but was downregulated in morphologically differentiated areas after 3 days in serum ( arrowhead ). ( n ) tnik expression was localized to the cytoplasm, and ( n, q ) expression appeared to be restricted to morphologically undifferentiated cells ( arrowhead ). ( o ) p130 cas was detected in a membrane / peripheral - cytoplasmic pattern in undifferentiated cells, ( r ) but this distribution was substantially altered in differentiating cells with a flattened morphology, which exhibited a general cytoplasmic, or perinuclear profile. scale bar for a - l: ( a, l ) 200 μm; ( c, d, f, h, i, j, k ) 100 μm; ( b, e, g ) 50 μm. scale bar for m - r: ( m, n, p, q ): 100 μm; ( o, r ): 50 μm. preliminary analyses also indicated that expression of some of these proteins was downregulated in differentiated cells, including p130 cas and the traf2 - and nck - interacting kinase ( tnik ). tnik is known to be involved in the inhibition of cell spreading via disrup tion of f - actin [ 34, 35 ]. immunoflu orescence was used to examine the expression of tnik and p130 cas during early differentiation of hescs. bg01 cultures were partially differentiated by growth in serum containing media for 3 days. this condition generated heterogeneous populations containing oct4 + cells with characteristic hesc morphology and less tightly packed, and morphologically differentiated areas, lacking expression of oct4 ( fig 4m, p ). tnik was expressed highly in undifferentiated hescs, and in the undifferentiated areas at day 3, but was downregulated in areas undergoing morphological differentiation ( fig 4n, q ). this may indicate that tnik is active in hescs and degraded rapidly upon differentiation. p130 cas was detected in a membrane / peripheral - cytoplasmic pattern in hescs ( fig 4o ). the distribution of p130cas was substantially altered in differentiating cells with a flattened morphology, exhibiting a general cytoplasmic, or perinuclear profile ( fig 4r ). this could indicate an alteration in the function of p130 cas as pluripotent cells differentiate. these analyses suggested that the change in expression or distribution of these proteins could be used as markers for undifferentiated hescs. comparison of proteomic and transcriptional profiles of hescswe have previously employed the illumina bead array system for the large - scale profiling of gene expression in hescs using 24, 000 transcript probes [ 11 ]. to compare proteomic and transcriptional analyses of hescs, the levels of > 600 proteins detected using large scale blotting were correlated with the levels of transcripts detected with the illumina platform ( additional file 3 ). in general, a close match between the expression level of transcript and protein was observed: transcripts for nearly all the detected proteins were also identified in the illumina analysis, and most proteins expressed at high levels also exhibited high mrna levels. we reasoned that a focused comparison of specific signaling pathways using a combination of proteomic and transcriptional data was likely to be much more informative than a global interrogation of hescs. several major signal pathways that have been suggested to be involved in self - renewal were examined to test this approach. these included the fgf, tgfβ, gsk3β / wnt / β - catenin and jak / stat pathways [ 17, 29, 36 - 39 ], as well as the more recently suggested mapk / erk and gap junction pathways [ 32, 40 ]. correlating transcriptional and proteomic data provided direct confirmation that these pathways were present and likely functional in hescs ( table 2 ). for example, fgf2 protein was expressed highly in hescs and expression of key members of the tgfβ, wnt, jak / stat and gap junction pathways, namely stat1, smads, gsk3β, β - catenin and connexin 43, were detected in both transcriptional and proteomic databases.