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. Author manuscript; available in PMC: 2015 Aug 13.
Published in final edited form as: Discoveries (Craiova). 2014 Jun 30;2(2):e16. doi: 10.15190/d.2014.8

Figure 1. Novel FOXO3 binding partners discovered by using a TAP-MS strategy.

Figure 1

A. Tandem Affinity Purification (TAP) followed by protein identification by Mass Spectrometry (MS) method was employed to identify novel FOXO3 binding partners. Lysates from HeLa cells transfected with either FOXO3-Flag-HA or EV-Flag-HA were immunoprecipitated with an anti-FLAG antibody, followed by a second purification with an anti-HA antibody. Denatured proteins (reducing Laemmli’s SDS-SB) were separated by SDS-PAGE. B. Proteins were stained with Silver stain. The bands corresponding to FOXO3 and PLK1 proteins identified by MS are presented. BSA (Bovine Serum Albumin) was used as a marker for protein quantity. C. Proteins identified by MS in the 60–65 kDa band presented in (B). PLK1 was one of the identified proteins (the arrow indicates PLK1). D. Most important/promising proteins identified after analyzing multiple bands by MS, covering 25–200 kDa gel range, are presented. The “Score” represents a probability Score determine with Sequest. EV – empty vector; MG132 was used to enrich for potential E3 ligases.