Stem Cells International / 2017 / Article / Fig 1

Research Article

The DEAD-Box RNA Helicase DDX3 Interacts with m6A RNA Demethylase ALKBH5

Figure 1

Identification of interaction between DDX3 and ALKBH5. (a) FLAG-DDX3 overexpression in HEK293T cells, relative DDX3 mRNA (upper panel), and protein levels (lower panel) are shown. (b) HA-ALKBH5 overexpression in HEK293T cells, ALKBH5 mRNA (upper panel), and protein levels (lower panel) are shown. (c) Overexpression of methyltransferases and demethylases in HEK293T cells, mRNA levels examined by qPCR. (d) Overexpression of the corresponding protein (indicated with arrowhead) examined by Western blots. (a–d) EV, empty vector; OE, overexpression. (e) DDX3 showed no interaction with the METTL3, METTL14, WTAP, or FTO, examined with IP and co-IP. (f) The interaction of ALKBH5 with DDX3 examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HeLa cells. (g) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with HA-ALKBH5 IP and FLAG-DDX3 co-IP in HEK293T cells. (h) The interaction of ALKBH5 with DDX3 in the presence or absence of RNase A, examined with FLAG-DDX3 IP and HA-ALKBH5 co-IP in HEK293T cells. (i) Endogenous DDX3 interacted with endogenous ALKBH5, examined with IP and co-IP; two known isoforms of ALKBH5 are indicated with arrowheads. IP and co-IP were performed in triplicates, and representative results are shown. ; values were determined with two-tailed Student’s t-test; error bars represent standard deviation (SD).
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)