Figure 4: Inflammatory stimuli increase PLCβ1 palmitoylation and its signaling activity.
(a) Increased palmitoylation was detected in endothelial cells after stimulation with SIRS plasma or inflammatory stimuli. Representative images of palmitoylation in cells treated with vehicle, histamine (10 μM, 15 min), thrombin (10 U ml−1, 30 min) or SIRS plasma (20% dilution, 1 h) in the absence or presence of 2-BP (pretreatment at 10 μM). Palmitoylated proteins were metabolically labelled with a palmitic acid analogue ω-alkynyl palmitic acid (Alk-C16). Labelled proteins were visualized by further probing with fluorescent azide via Click chemistry reaction. Palmitoylated proteins are shown in green and nuclei blue. Scale bar, 30 μm. Bar graph shows quantification of total palmitoylation signal intensity. Results are from three independent experiments presented as mean±s.e.m. *P<0.05 versus control+vehicle, #P<0.05 versus stimulus+vehicle. (b,c) The levels of palmitoylated PLCβ1 in endothelial cells were determined using two different methods. (b) Acyl-biotin exchange. Whole cell lysates from thrombin (10 U ml−1) or vehicle treated ECs, with or without 2-BP, were subject to acyl-biotin exchange to isolate palmitoylated proteins in the presence of hydroxylamine (NH2OH). The samples were then used in Western blotting for PLCβ1. Band intensity was quantified and normalized to control. Results represent three independent experiments. *P<0.05 versus vehicle+control, #P<0.05 versus vehicle+thrombin. (c) Pull-down of palmitoylated proteins and immunoblotting for PLCβ1. ECs were incubated with Alk-C16 overnight and then treated with histamine (10 μM) or vehicle. Whole cell lysates were collected, and palmitoylated proteins were pulled down using Azide Agarose Resin via Click chemistry reaction. Palmitoylated protein samples were subject to Western blotting for PLCβ1. Band intensity was quantified and normalized to control group. Bar graph represents four independent experiments. *P<0.05 versus vehicle. (d) IP3 levels in ECs subjected to histamine stimulation (10 μM) with or without 2-BP (10 μM). Bar graph represents mean±s.e.m. from three independent experiments. *P<0.05 versus vehicle, #P<0.05 versus vehicle+histamine.
