Verification of protein expression using immunocytochemistry. (A-K) Ten proteins that were detected in undifferentiated hESCs by western blotting were also detected by immunofluorescence of BG01 cells grown in MEF-CM. Ras-GAP (pY460) is a phosphorylated form of Ras-GAP. The same antibodies 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.