Figure 2: Trim71 interacts with Argonaute2. | Nature Communications

Figure 2: Trim71 interacts with Argonaute2.

From: Trim71 cooperates with microRNAs to repress Cdkn1a expression and promote embryonic stem cell proliferation

Figure 2

(a) Cell extracts were prepared from KH2 ESCs expressing either Flag-Lin28 or Flag-Trim71. Flag-affinity purifications were washed with buffer containing the indicated KCl concentration. Where indicated, RNase A was added to the lysates for IP. The input and affinity eluates were analysed by western blot. (b) Analysis of miRNA and Argonaute protein in RNase-treated extracts. TaqMan qRT–PCR for the indicated miRNA (Top), Error bars represent mean+/−s.d. with n=3, and western blot for Ago2 (bottom). (c) Flag-affinity purifications on whole-cell extracts from HEK293 (left) and P19 (right) that were transfected to express Flag-Trim71 or the indicated control proteins. Western blots performed using α-Flag and α-Ago2 antibodies. (d) Flag IPs performed as in (a) (100 mM KCl washes). The eluates were analysed by western blot. (e,f) Endogenous co- immunoprecipitation from J1 whole-cell extracts performed with the indicated antibodies. The immunoprecipitated proteins are indicated by arrowheads.

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