bamhi ggg met bira f  (Addgene inc)

Journal: Cells

Article Title: Disclosing the Interactome of Leukemogenic NUP98-HOXA9 and SET-NUP214 Fusion Proteins Using a Proteomic Approach

doi: 10.3390/cells9071666

Figure Lengend Snippet: Proximity-dependent biotin identification (BioID) fusion protein expression and biotinylation of endogenous proteins. ( A ) Localization of NHA9-BioID, SN214-BioID, and BirA R118G was evaluated by immunostaining with anti-HA antibody. ( B ) Localization of GFP-tagged NUP98-HOXA9 and SET-NUP214 was evaluated by green fluorescent protein (GFP) fluorescence. NHA9-BioID and SN214-BioID exhibit the same distribution pattern in nuclear foci and at the nuclear envelope as their GFP-tagged counterparts. ( C ) Detection of protein biotinylation by Streptavidin-488. Cells were transfected with NHA9-BioID, SN214-BioID, and BirA R118G and probed with Streptavidin-Alexa Fluor™ 488 conjugate. Shown are representative confocal images. DNA was visualized with DAPI (blue). Scale bars, 10 µm. ( D ) For detection of protein biotinylation by NHA9-BioID, SN214-BioID, and BirA R118G , corresponding cell lysates were enriched on Streptavidin-coated magnetic beads and whole protein lysates and the bound fractions were analyzed by immunoblotting. Note, virtually no specific bands were detected in the bound fraction of BirA R118G , in contrast to the bound fractions of NHA9-BioID and SN214-BioID, which exhibited patterns of differentially biotinylated proteins.

Article Snippet: pcDNA3.1 MCS-BirA(R118G)-HA was a gift from Dr. Kyle Roux (Addgene plasmid # 36047; [ ]) and BirA(R118G)-HA destination vector from Dr. Karl Kramer (Addgene plasmid # 53581).

Techniques: Expressing, Immunostaining, Fluorescence, Transfection, Magnetic Beads, Western Blot