FIGURE 2.

Structural behavior of GFP fusion proteins. Extracts of HeLa cells expressing GFP fusion proteins were analyzed on a Superose 6 HR 10/30 column equilibrated in 20 mm HEPES, pH 7.5, 200 mm NaCl, 2 mm dithiothreitol, and 0.001% Tween 20 and developed at a flow rate of 0.2 ml/min. Fractions of 0.5 ml were collected and analyzed by Western blotting with antibodies directed to GFP. A typical elution profile is shown (absorbance at 280 nm, thick line). The column was previously calibrated with bovine serum albumin (thin line); the arrow shows the elution volume of purified MARS. Extracts of stable HeLa cells expressing MetRS-GFP, ΔNMetRSΔC-GFP, or p43-GFP-St or from HeLa cells that transiently expressed p43-GFP-Tr were analyzed independently, and the fractions were probed with anti-GFP antibodies.