Figure 4. Dephosphorylation of Sec31 at Serines 527 and 799 increases its affinity to Sec23.

(A) HEK293 cells were co-transfected with indicated FLAG-tagged Sec31 mutants and GFP-tagged Sec23. Sec23 was immunoprecipitated with anti-GFP beads and subjected to western blotting with anti-FLAG and anti-GFP antibodies. The “input 10% Sec31” is a western blot of 10% aliquots of total cell lysates. The normalized ratio of Sec31 bound to Sec23 was shown below. W985A is a known Sec23-binding defect mutant [52]. The most right lane is a negative control with GFP. The graph below the blots shows the average of the quantification of three independent experiments with SD. (B) FLAG-Sec31 and GFP-Sec13 (instead of GFP-Sec23) were co-expressed and their interactions detected by co-immunoprecipitation and western blotting following the procedures described in Figure 4A. The lane on right most is a negative control with GFP.