Fig 4. Glycogenic activity of different mutated forms of R6.

(A) Measurement of glycogenic activity of different R6 mutated forms. N2a cells were transfected using 1 μg of pFLAG plasmid (negative control), pFLAG-R6 plasmid or its corresponding mutants. Forty-eight hours after transfection, the amount of glycogen was determined as described in Materials and Methods and represented as μg of glucose/mg of protein/relative amount of R6 respect to actin (wild type value considered as 1). Bars indicate standard deviation of three independent experiments (**p<0.01 or ***p<0.001, compared with control cells transfected with an empty plasmid; ##p<0.01, compared with cells expressing R6-WT). An inset with the mean values +/- standard deviation is included. (B) Protein levels of FLAG-R6 forms. A representative western blot analysis is shown. Cell extracts (30 μg) were analyzed using the corresponding anti-FLAG and anti-actin antibodies.