MOESM6 of DUF3669, a “domain of unknown function” within ZNF746 and ZNF777, oligomerizes and contributes to transcriptional repression
2019-12-20T06:34:47Z (GMT) by
Additional file 6 In vitro expression of DUF3669-containing polypeptides derived from ZNF746 and ZNF777. SoluBL21 (DE3) E. coli were transformed with 10 ng of the indicated prokaryotic constructs. The protein expression was induced by adding 0.1 mM IPTG to the bacterial suspensions at OD600 = 0.4–0.6. Bacterial cell pellets were lysed with 1 x SDS sample buffer (ni = non-induced bacteria, 25 μl total extract; i = bacteria after 4 h induction with IPTG, 25 μl). Soluble crude fractions of recombinant proteins were obtained from induced bacteria that were washed with 1x ice-cooled PBS, re-suspended in lysis buffer and lysed by sonication. Different amounts of bacterial lysates were loaded to the SDS-polyacrylamide gels (L1: 4 μl, L2:8μl, L3:20μl). Extracts were subjected to Western blotting and the membranes probed with polyclonal anti-GST and monoclonal anti-His tag (a, two-color results shown as one black/white overlay representation), polyclonal anti-MBP (b) or monoclonal anti-His tag (c) antibodies.