Fig. 2.

GULP1 interacts with ATG14. A Bacterially expressed GST and GST-GULP1 were used as baits for pull-down assay from ATG14, Beclin-1, VPS15 and VPS34 transfected cell lysate respectively. Overexpressed proteins in lysate and pull-downs were analyzed by immunoblotting. B Coimmunoprecipitation was performed from HEK293 cells transfected with ATG14 or ATG14 + GULP1. ATG14 in cell lysate was immunoprecipitated using a mouse anti-FLAG antibody. GULP1 and ATG14 in lysate and immunoprecipitates were analyzed by immunoblotting with anti-GUP1 G-R3 and anti-FLAG 20543-1-AP. C ATG14 in rat brain lysate was immunoprecipitated by an anti-ATG14 antibody. ATG14, GULP1, Beclin1, Vps34 and Vps15 in lysate and immunoprecipitate were immunoblotted with anti-ATG14 PD026, anti-GULP1 G-R3, anti-Beclin1 Bec-R3, anti-Vps34 F-11and anti-Vps15 JK-13. D In a fluorescent PLA assay, PLA signals representing GULP1-ATG14 interaction were detected in GULP1 + ATG14 transfected cells. Representative images are shown. Data were obtained from at least 60 cells per transfection and the experiments were repeated 3 times. Error bars are sem. ***p < 0.001. No-antibody (No Ab), anti-FLAG, and anti-GULP1 control PLAs were performed. E Bacterially expressed His₆-ATG14 was used as baits to pull down purified GST or GST-GULP1. GST proteins were probed with a rat anti-GST serum. Left panel shows the purified protein used for pull-down. F Bacterially expressed GST and GST-GULP1 fragments were used as baits for pull-down assay from ATG14 transfected cell lysate. ATG14 in lysate and immunoprecipitates were immunoblotted with anti-ATG14 19,491-1-AP. Bottom panel: Coomassie Blue staining of GST-baits used. G Bacterially expressed His₆-ATG14 was used as baits for pull-down assay from GST and GST-GULP1 fragments transfected cell lysate. GST proteins in lysate and immunoprecipitates were analyzed by immunoblotting. Lower panel: Coomassie Blue staining of His₆-ATG14 baits. H Cells were co-transfected with GULP1 or GULP1 K66A/K69A (GULP1m). GST-fused ATG14 fragment was used as bait to pull down GULP1 in transfected cell lysates. GST-fusion proteins and GULP1 in lysates and pull-downs were probed with rat anti-GST serum and anti-GULP1 G-R3 respectively. Bar chart shows the densitometric quantification of relative GULP1 levels in pulldowns. **p < 0.01. I Cells were co-transfected with ATG14 + GULP1 or ATG14 + GULP1 K66A/K69A (GULP1m). ATG14 in cell lysate was immunoprecipitated using anti-FLAG M2 antibody. GULP1 and ATG14 in lysate and immunoprecipitates were immunoblotted with rat anti-GULP1 serum and anti-FLAG 20543-1-AP respectively. Bar chart shows the densitometric quantification of relative GULP1 levels in IPs. ***p < 0.001