Analysis of Familial Hemophagocytic Lymphohistiocytosis Type 4 (FHL-4) Mutant Proteins Reveals that S-Acylation Is Required for the Function of Syntaxin 11 in Natural Killer Cells
Figure 9
Syntaxin 11 is S-acylated and this is dependent on cysteines in the C-terminal region.
(A) Acyl-biotinyl exchange. Free cysteines are blocked with the alkylating agent NEM, long chain fatty acid groups are cleaved from proteins using hydroxylamine revealing free cysteines, which are biotinoylated, enabling proteins to be pulled down with avidin beads. Non-transfected YTS cells (B) and YTS cells transfected with either GFP-syntaxin 11, GFP-syntaxin 11 Q268X or GFP-syntaxin 11C5A (C) were analysed with acyl-biotinyl exchange. Precipitated proteins from samples that had been incubated in the absence (negative control) or presence of hydroxylamine (S-acylated fraction) were resolved by SDS-PAGE. Syntaxin 11 and GFP-syntaxin 11 fusions were detected by probing immunoblots with syntaxin 11 specific antibodies. The non-S-acylated protein calreticulin served as a negative control and was detected with a rabbit anti-calreticulin antibody.