Fig. 3
Activation and function of PTE in response to TNF. a APT1 and APT2 are part of the TNF-RSC: TNF-R1 was activated, lysed in the presence of detergents and precipitated using Fc60TNF, activating only TNF-R1. Panel one: TNF-R1 displaying the reported increase in K63-ubiquitination in response to TNF [5]. Panel two: Constitutive TNF-RSC:APT1 interaction, decreasing at 10 min. Panel three: Transient recruitment of APT2 to the TNF-RSC. Panels 4–6 show WB with 10 μg of input lysate. b Activation of endogenous S-depalmitoylases in response to TNF, analyzed by imaging flow cytometry using fluorescent probes: DPP-2 for global S-depalmitoylation and DPP-3 for APT1 activity. Left panel one: Representative images of inactive APT2. Left panel two: Shows cells with activated fluorescent probe (DPP-2; green) in response to TNF. PM is stained in red. Panel three: histograms representing enzyme activity. Dashed black: untreated cells. Green: addition of DPP-2 and TNF to cells kept on ice. Addition of DPP-2/TNF at 37 °C for 5 min (red), 10 min (orange), 15 min (blue). Black: DPP-2/Palmostatin B (pan PTE inhibitor). Dashed red: DPP-2/ML349 (APT2 selective inhibitor). Right panels: DPP-3 is not activated in response to TNF (same order and color code as A). c Left panel: representative WB from acylRAC probed for TNF-R1 at the conditions: no TNF (ctrl), 30 min TNF, ML349 with 30 min TNF, and ML349. PPT-1 serves as loading control. Right panel: WB quantification by densitometry. WB loading control, see Additional file 3: Figure S3. d His6rAPT2 was expressed in and affinity purified from E. coli. WB was probed using APT2 and His antibodies. rAPT2 activity after 20 min incubation at the conditions: DPP-2 (light grey), DPP-2 + APT2 (black), DPP-2 + APT2 + Palmostatin B (medium grey), DPP-2 + Palmostatin B (dark grey). e WB of input and acylRAC +/− rAPT2 (30 μM, 1 h 37 °C): Palmitoylation of TNF-R1 decreases while CD71, integrin α6, PPT-1 and Rab5 are not affected. The lower panel shows quantification of the WB for TNF-R1 normalized to PPT-1 levels