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. Author manuscript; available in PMC: 2013 Mar 21.
Published in final edited form as: J Am Chem Soc. 2012 Mar 7;134(11):5149–5156. doi: 10.1021/ja209868g

Figure 2. Analysis of cells expressing mbGluc-biotin reporter.

Figure 2

(a) Gli36-sshBirA cells were transduced with the lentivirus vector expressing mbGluc-biotin or GFP control. Forty-eight hours later, cells were lysed and lysates were analyzed by Western blotting for both biotinylation efficiency and Gluc expression using either streptavidin-HRP conjugate or a combination of Gluc-specific antibody followed by secondary-HRP conjugate respectively. Blots were also analyzed for β-tubulin to show equal loading. (b) Gli36 cells expressing either mbGluc-biotin or native secreted Gluc were imaged for Gluc expression on the cell surface by adding coelenterazine directly to viable cells followed by photon count using a luminometer. Data presented as average ± standard deviation (SD). (c) Viable Gli36-sshBirA cells infected with lentivirus vector encoding mbGluc-biotin and GFP or GFP alone were labeled with anti-biotin antibody conjugated to APC or Gluc antibody followed by a secondary-Alexa647 conjugate and analyzed by FACS. (d) Gli36-sshBirA cells expressing mbGluc-biotin and GFP were plated on coverslips and stained with either streptavadin-Alexa647 for biotin presentation on the cell surface (bottom pannel) or with a combination of Gluc antibody followed by secondary-Alexa647 conjugate (top pannel). Cells were then fixed using 4% paraformaldehyde and analyzed by fluorescence microscopy for both GFP expression and biotin or Gluc staining.