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Fig. 1
Generation and characterization of ESCs in which Fam40b was constitutively knocked down (KD) by transfection with pGFP-V-RS, expressing shRNA directed against the Fam40b RNA, the GFP and the puromycin resistance cassette. (a) Fluorescence microscopy of control 12-day EBs derived from ESCs transfected with the pGFP-V-RS vector without shRNA (control) and EBs derived from ESCs transfected with the pGFP-V-RS shRNA expressing vector containing 29-mers shRNA (Fam40b KD EBs) (scale bar: 50μm). (b) Semiquantitative RT-PCR analysis of the expression of GFP and Fam40b in undifferentiated control and KD Fam40bESCs as well as in 4-, 8- and 12-day control and KD EBs generated by the hanging drop protocol. (c) Expression of the FAM40B protein during differentiation of WT ESCs (upper panel) and during differentiation of Fam40B KD ESCs. Detection has been performed with FAM40b primary antibody (sc-162799, Santa Cruz Biotechnology; 1:500 dilution) and with the secondary donkey anti-Goat IgG antibody (1:10000 dilution). GAPDH has been detected using the anti-GAPDH antibody (1:25000 dilution). (d) Densitometric analysis of the FAM40B and the corresponding GAPDH bands densities has been performed by using the ImageJ 1.47v software (National Institutes of Health, Bethesda, MD, USA) and the ratio of the FAM40B/GAPDH band densities has been blotted for the different time points of differentiation