Figure 7. The cellular behavior of... | Rockefeller University Press

Figure 7.

$Figure 7. The cellular behavior of KLHL12 is recapitulated in the cell-free reaction. (A) Budding reactions were supplemented with 2 µg/µl 293T cytosol containing FLAG-tagged KLHL12 wild-type or FG289AA mutant in addition to 2 µg/µl HT1080 cytosol. Cytosol (293T cells) that contained GFP and 4 µg/µl HT1080 cytosol were used as controls with endogenous levels of wild-type KLHL12. Top fractions were collected after density gradient flotation for immunoblotting analyses. A representative experiment of seven independent repeats. (B) Relative enrichment of KLHL12 detected in the floated fraction. Densitometry quantification of FLAG intensities in the floated fraction was divided by the corresponding FLAG intensity in the 7,000-g supernatant (input for flotation). Error bars = SD. Paired t test. ***, P < 0.0001. n = 7. Mean of wild-type/mean of FG289AA = 6.80. (C) Relative budding efficiency of PC1, HSP47, ERGIC53, and SEC22B in reactions that were supplemented with 293T cytosol that contained wild-type KLHL12 or KLHL12 FG289AA. Vesicle budding efficiencies were calculated from densitometry quantification relative to the control lane, where 293T cytosol that contained GFP was supplemented. Paired t test was used to analyze the difference between wild-type and FG289AA samples of each cargo; p-values were 0.0412 for PC1 (*, P < 0.05), 0.1215 for HSP47, 0.5307 for ERGIC53, and 0.2993 for SEC22B. Error bars = SD. n = 7.$

The cellular behavior of KLHL12 is recapitulated in the cell-free reaction. (A) Budding reactions were supplemented with 2 µg/µl 293T cytosol containing FLAG-tagged KLHL12 wild-type or FG289AA mutant in addition to 2 µg/µl HT1080 cytosol. Cytosol (293T cells) that contained GFP and 4 µg/µl HT1080 cytosol were used as controls with endogenous levels of wild-type KLHL12. Top fractions were collected after density gradient flotation for immunoblotting analyses. A representative experiment of seven independent repeats. (B) Relative enrichment of KLHL12 detected in the floated fraction. Densitometry quantification of FLAG intensities in the floated fraction was divided by the corresponding FLAG intensity in the 7,000-g supernatant (input for flotation). Error bars = SD. Paired t test. ***, P < 0.0001. n = 7. Mean of wild-type/mean of FG289AA = 6.80. (C) Relative budding efficiency of PC1, HSP47, ERGIC53, and SEC22B in reactions that were supplemented with 293T cytosol that contained wild-type KLHL12 or KLHL12 FG289AA. Vesicle budding efficiencies were calculated from densitometry quantification relative to the control lane, where 293T cytosol that contained GFP was supplemented. Paired t test was used to analyze the difference between wild-type and FG289AA samples of each cargo; p-values were 0.0412 for PC1 (*, P < 0.05), 0.1215 for HSP47, 0.5307 for ERGIC53, and 0.2993 for SEC22B. Error bars = SD. n = 7.