381824.fig.003a
(a)
381824.fig.003b
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381824.fig.003c
(c)
Figure 3: FLAG purifications. (a) Western blot analysis of FLAG-purified M7 nuclear and cytosol extracts using TAP negative MFVD cells as control. Compared are SRC1, beta catenin (CTNNB1), and AR expression in extracts before (NE/CE) and after (FLOW) purification and in the TEV-eluted fractions. AR (WT and N-TAP-mAR comigrate) is recovered in TEV eluate from the M7 but not from the MFVD control purifications. (b) Western blot analysis of FLAG-purified P17 nuclear and cytosol extracts using TAP negative PC1 cells as control. Compared are SRC1, beta catenin, and AR expression in extracts before (NE/CE) and after (FLOW) purification and in the TEV-eluted fractions. AR (WT and N-TAP-mAR are here distinguishable) is recovered in TEV eluate from the P17 but not from the PC1 control purifications. (c) Coomassie stained 5% PAGE loaded with TEV-eluted fractions from the control cell line PC1 and the NTAP-mAR expressing cell line P17 grown at 33°C and 37°C after FLAG purification. This gel was prepared for cutting out gel slices (1–7), which were then submitted for mass spectrometry. The arrows indicate the N-TAP-mAR migration. M is the size estimate by a prestained protein ladder in kilo Daltons (kDa).