Fig. 6

Activation of PKA and increased Arf1-GTP level plays a crucial role in KDELR relocalization. A Effect of the co-expression of KDEL-cargo (hGH-GFP-myc-KDEL) on KDELR1-mCherry co-immunoprecipitation in HeLa cells. HeLa cells co-transfected KDELR1-mCherry with hGH-GFP-myc or hGH-GFP-myc-KDEL were firstly treated with DSP crosslinker in PBS at 37 °C for 30 min. Cells were lysed and the extracts were immunoprecipitated with anti-RFP agarose beads. B Effect of ACBD3-KO on KDELR1-mCherry co-immunoprecipitation in HeLa cells. HeLa-WT or ACBD3-KO cells transfected with KDELR1-mCherry (mCherry-SNX3 transfection as a control) were lysed and immunoprecipitated with anti-RFP agarose beads. C Both PKA-Cα-EGFP and PKA-RII-EGFP co-immunoprecipitate with ACBD3 in HeLa cells. D Effect of ACBD3-KD/KO or ACBD3 over-expression on the luciferase expression in HeLa cells, expressing the CRE-controlled luciferase. HeLa-WT, ACBD3 stable knockdown, ACBD3-KO, or EGFP-ACBD3 stably over-expressing cells seeded in 24-well plate were transfected with pCRE-luciferase plasmid. Cells were then lysed, and equal protein amounts of lysate per condition were assayed using a luciferase assay system. Statistical analysis was performed using one-way ANOVA with a Tukey’s post hoc test (mean±SD, *p < 0.05; **p < 0.01; ****p < 0.0001). E HeLa WT, ACBD3-KD, or ACBD3-KO cells were analyzed for phosphorylation levels of Src family. F PKA inhibition by H89 decreases ARF1-GTP level in ACBD3-KO HeLa cells. HeLa-WT or ACBD3-KO cells were firstly treated with H89 for 2 h and then analyzed for levels of Arf1-GTP. Equal aliquots of lysates were analyzed for Arf1-GTP by using ARF1 activation assay kit utilizes pull-down with GST-GGA3. G, H Confocal images of KDELR1^endo-3xFlag-mCherry in HeLa cells showing that PKA inhibition by H89 does not result in re-distribution of KDELR1^endo-3xFlag-mCherry to the ER, while PKA activation by 8-Bromo-cAMP results in re-distribution of KDELR1^endo-3xFlag-mCherry to the ER in vivo without affecting ManII localization. I Confocal images of KDELR1^endo-3xFlag-mCherry in HeLa cells showing that only over-expression of PKA-Cα-EGFP results in re-distribution of KDELR1^endo-3xFlag-mCherry to the ER in vivo. Scale bars = 10 μm