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letter
. 2018 Mar 14;184(5):876–881. doi: 10.1111/bjh.15189

Figure 1.

Figure 1

GFP‐p60‐BBF2H7 over‐expression in primary human erythroblasts and up‐regulation of endogenous SEC23A. (A) Cloning of the N‐terminal p60‐fragment of BBF2H7 in a GFP lentiviral vector (GFP‐p60‐BBF2H7). p60 encompasses the first 377 residues of BBF2H7 (human BBF2H7 numbering). The last 3 amino acids of the N‐terminal GFP are shown in green, the vector sequence between the GFP and p60 BBF2H7 is shown in black (5 amino acids: GGSVD) and the first 3 amino acids of p60‐BBF2H7 are shown in red. (B) Protocol and experimental timeline followed for all cultures. (C–D) Western blotting of primary human erythroblast lysates from 3 separate cultures, all derived from the same healthy control donor (un‐transduced, transduced with GFP or GFP‐p60 BBF2H7) using antibodies against BBF2H7 (C, left), GFP (C, right), SEC23A (D, left), SEC23B (D, right) or actin as a loading control (C‐D). The anti‐SEC23A and anti‐SEC23B antibodies have been described previously (Satchwell et al, 2013). (E–F) Western blotting of 3 separate cultures (un‐transduced, transduced with GFP or GFP‐p60‐BBF2H7) of primary human erythroblasts lysates all derived from the same CDAII patient [E109K/E109K (culture 1)], using antibodies against BBF2H7 (E), SEC23A (F, left; the arrow points to SEC23A, the asterisks indicate non‐specific bands), SEC23B (F, right) or actin as a loading control. (G) Quantification by densitometry of SEC23A expression on Day 10 from Western blots of healthy control cultures (n = 3) and CDAII patients (4 cultures from 3 patients). The y axis values show the ratio of SEC23A over actin, normalised to that of the GFP cultures. For each culture, the value obtained for the GFP cells was set to 1 and the values for the GFP‐p60 BBF2H7 cells show the fold increase when compared to GFP expressing cells from the same donor. The values obtained for the 3 control cultures expressing GFP‐p60 BBF2H7 are shown in different shades of grey. The values obtained for 4 CDAII cultures expressing GFP‐p60 BBF2H7 are for E109K/E109K (culture 1, dark blue); E109K/E109K (culture 2, light blue); R190X/S603L (purple) and R14W/R554X (light green).