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. 2015 Jul 31;290(37):22543–22557. doi: 10.1074/jbc.M115.669408

FIGURE 6.

FIGURE 6.

P222L mutation affects TRBP-PACT and PACT-PACT interactions. A, co-immunoprecipitation of FLAG-PACT with myc-TRBP at early time point after ER stress. HeLa cells were transfected with either FLAG- WT PACT or FLAG-P222L mutant and myc-TRBP. 24 h after transfection cells were either left untreated or treated with 50 μg/ml tunicamycin. 2 h after treatment cells were harvested, FLAG-PACT was immunoprecipitated using anti-FLAG mAb-Sepharose, and co-immunoprecipitation of myc-TRBP was analyzed by Western blot analysis with anti-myc mAb (myc-TRBP panel). The blot was stripped and re-probed with anti-FLAG mAb to ascertain an equal amount of FLAG-PACT protein in each lane (FLAG-PACT panel). Input blot: Western blot analysis of total proteins in the extract with anti-myc mAb. B, co-immunoprecipitation of FLAG-PACT with myc-TRBP at late time points after ER stress. HeLa cells were transfected with either FLAG-WT PACT or FLAG-P222L mutant and myc-TRBP. 24 h after transfection, cells were either left untreated or treated with 50 μg/ml tunicamycin. AT 8 and 24 h after tunicamycin treatment, cells were harvested, FLAG-PACT was immunoprecipitated using anti-FLAG mAb-Sepharose, and co-immunoprecipitation of myc-TRBP was analyzed by Western blot analysis with anti-myc mAb (myc-TRBP panel). The blot was stripped and re-probed with anti-FLAG mAb to ascertain an equal amount of FLAG-PACT protein in each lane (FLAG-PACT panel). Input blot: Western blot analysis of total proteins in the extract with anti-myc mAb. C, co-immunoprecipitation of FLAG-PACT and myc-PACT. HeLa cells were transfected with either FLAG-WT PACT and myc-WT PACT or FLAG-P222L mutant and myc-P222L mutant. 24 h after transfection, cells were either left untreated or treated with 50 μg/ml tunicamycin. 2 h after treatment, cells were harvested, FLAG-PACT was immunoprecipitated using anti-FLAG mAb-Sepharose, and co-immunoprecipitation of myc-PACT was analyzed by Western blot analysis with anti-myc mAb (myc-PACT panel). The blot was stripped and re-probed with anti-FLAG mAb to ascertain an equal amount of FLAG-PACT protein in each lane (FLAG-PACT panel). Input blot: Western blot analysis of total proteins in the extract with anti-myc mAb. D, mammalian two-hybrid assay. COS-1 cells were transfected with 250 ng of each of the two test plasmids encoding proteins to be tested for interaction, 50 ng of the reporter plasmid pG5Luc, and 1 ng of plasmid pRL-Null to normalize transfection efficiency. Cells were harvested 24 h after transfection, and cell extracts were assayed for luciferase activity. The plasmid combinations are as indicated. C1, WT PACT/GAL4DBD and VP16AD EV (negative control); C2, GAL4DBD EV and WT PACT/VP16AD (negative control); WT/WT, WT PACT/GAL4DBD and WT PACT/VP16AD; C3, P222L/GAL4DBD and VP16AD EV (negative control); C4, GAL4DBD EV and P222L/VP16AD (negative control); mut/mut, P222L/GAL4DBD and P222L/VP16AD. The experiment was repeated twice with each sample in triplicate, and the averages with S.E. bars are presented. Student's t tests performed to calculate p values indicated that the differences observed between the negative controls and test values (C1, C2 and WT/WT, C3, C4 and mut/mut) were highly significant: bracket * (0.0071), bracket ** (0.0001), bracket # (0.0037), bracket ## (0.0002). The difference between WT/WT and mut/mut interaction was also highly significant as indicated by the p value represented by bracket ### (0.0001). RLU, relative light units. E, Western blot analysis. COS-1 cell extracts were examined by Western blot analysis using an anti-GAL4-DBD mAb (Santa Cruz), anti-VP16AD Ab (Santa Cruz), and anti-β-actin mAb. The samples are indicated on top of the lanes.