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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: J Cell Biochem. 2019 Jun 27;120(11):19004–19018. doi: 10.1002/jcb.29223

Figure 3: Effect of FS mutation on interaction with PKR and PACT.

Figure 3:

(A, C, and E) Pull down assay of in vitro translated proteins with pure recombinant PACT, PKR, and FS proteins. 5 μl of in vitro translated, 35S-labeled flag-tagged PKR (A) or PACT (C) or FS (E) protein was mixed with 100 ng of purified, hexahistidine tagged recombinant PKR, PACT, and FS proteins immobilized in Ni-charged Sepharose beads. Pull down of 35S-labelled PKR, PACT and FS was analyzed by SDS-PAGE after washing the beads five times with wash buffer. Total lane: total input PKR, PACT, and FS as indicated (50% of the bound samples); Bound lanes: the pulled down PKR (A) or PACT (C) or PKR, PACT and FS (E). B, D, and F. Quantification of data in 3 A, 3 C, and 3E. The radioactivity present in the bands was measured by phosphorimager analysis and the % pull down was calculated as (radioactivity present in the pull down (bound) PKR, PACT, FS bands/the radioactivity present in the PKR, PACT, FS bands in the total lane) X 100. Error bars: standard error of mean from 4 independent experiments. Student T-tests were performed, and p values are as follows (B) * = 0.00018, ** = 0.00013, (D) * = 0.00019, and ** = 0.00009, (F) * = 0.00002 and ** = 0.00001, n=4.