Fig. 5

Palmitoylation inhibition mediated by the local anesthetics resulted in the disappearance of GP130 in the membrane fractions. a ABE was performed on proteins from U251 GSCs transfected with ZDHHC15 shRNA or treated with 2BP (50 μM) or PalmB (1 μM) for 48 h. The presence or absence of hydroxylamine (HAM) during the reaction was used as a control for reaction specificity. Western blot analysis with streptavidin-horseradish peroxidase is shown depicting the banding pattern of S-acylated proteins from 1 or 3 mg of total protein. b Protein accumulation (detected by western blot analysis) and palmitoylation level (detected by ABE) in GSCs treated with or without prilocaine, lidocaine, procaine, and ropivacaine at different concentrations (5 μM, 10 μM, and 20 μM, respectively). β-Actin was used as a loading control. c Expression of p-STAT3 (Y705) in U251 GSCs (monolayer culture) transfected with ZDHHC5 shRNA and treated with prilocaine, lidocaine, procaine, and ropivacaine (20 μM) for 48 h was analyzed by immunofluorescence staining. Scale bar, 100 μm. d The expression of GP130 in U251 GSCs (monolayer culture) transfected with ZDHHC5 shRNA and treated with prilocaine, lidocaine, procaine, and ropivacaine (20 μM) for 48 h was analyzed by immunofluorescence staining. Scale bar, 100 μm. e GSCs were transfected with ZDHHC5 shRNA or treated with prilocaine, lidocaine, procaine, and ropivacaine (20 μM) and harvested after 48 h. Cellular fractionation was performed to separate cytosolic and membrane fractions. Fractionates were then subjected to western blot analysis to detect the distribution of GP130