Extended Data Fig. 1: STAT3 is S-palmitoylated at evolutionarily conserved cysteine residues and mutation of STAT3 palmitoylation sites does not affect disulfide formation and protein stability.

a, HEK293A cells were incubated for 4 h with DMSO or 50 μM clickable probes. Cell lysates were reacted with biotin azide for enrichment of labelled proteins with streptavidin beads and identified by mass spectrometry. The peptide spectral counts are shown in the table. b, HEK293A cells were labelled with 50 μM chemical probe (alk-C₁₆) for 4 h. Cell lysates were reacted with biotin azide and precipitated with streptavidin beads, and subjected to western blot using anti-STAT3 antibody. Endogenous STAT3 palmitoylation was analysed by western blot. c, HEK293A cells were transfected with MYC–STAT3 wild type and labelled with 50 μM chemical probe (alk-C₁₆) for 4 h. Cell lysates were subjected to streptavidin blot, after anti-MYC immunoprecipitation and subsequent click reaction. d, HEK293A cells were transfected with empty vector or GFP–STAT1 and labelled with 50 μM chemical probe (alk-C₁₆) for 4 h. After click reaction, cell lysates were subjected to streptavidin blot, showing detection of STAT1. e, Analysis similar to that in c was performed in HEK293A cells transfected with Flag–STAT1α and Flag–STAT1β. f, HEK293A cells were transfected with Flag–STAT3 or Flag–STAT5 and labelled with 50 μM chemical probe (alk-C₁₆) for 4 h. Cell lysates were reacted with biotin azide and precipitated with streptavidin beads, and subjected to western blot using anti-Flag antibody. g, h, Analysis similar to that in e was performed in HEK293A cells transfected with Flag–STAT3, Flag–STAT2, Flag–STAT4 or Flag–STAT6. i, HEK293A cells were pulse-labelled with 50 μM chemical probe (alk-C₁₆) for 4 h, and subsequently chased by the addition of an excess of 50 μM BSA-conjugated palmitic acid. Cells were collected at the indicated time point, and subjected to analysis of STAT3 palmitoylation. j, Schematic of Flag–STAT3 and Flag–STAT3 truncation-mutant constructs. k, HEK293A cells were transfected with vector control, Flag-tagged wild-type STAT3, STAT3(ΔSH2), STAT3(1–585) or STAT3(586–770) mutant, and labelled with 50 μM chemical probe (alk-C₁₆) for 4 h. STAT3 palmitoylation levels were analysed by click reaction and streptavidin bead pulldown, followed by western blotting using anti-Flag antibody. l, Alignment of STAT3 protein sequence among different species. m, HEK293A cells were transfected with Flag-tagged wild-type STAT3, or the STAT3(C687S), STAT3(C712S) or STAT3(C687S/C712S) (here labelled 2CS) mutants, and total cell lysates were subjected to APE assay. Samples were analysed by western blot using anti-Flag antibody. The top band indicates the palmitoylated STAT3. n, The palmitoylated STAT3 in m was quantified using ImageJ. The data are presented as mean ± s.e.m., n = 3 biologically independent experiments. P value was determined by two-tailed t-test. o, HEK293A cells were transfected with Flag-tagged wild-type STAT3, or the STAT3(C687S), STAT3(C712S) or STAT3(C687S/C712S) mutants, and total cell lysates were reduced with DTT. Samples were analysed by western blot using anti-Flag antibody. (p) HEK293A cells were transfected with Flag-tagged wild-type STAT3 or STAT3(C687S/C712S) mutant, and free thiols of protein were blocked by NEM. Disulfide bonds were reduced by DTT. Maleimide–PEG was applied to attach at the reduced thiols. Samples were analysed by western blot using anti-Flag antibody. q, HEK293A cells were transfected with Flag-tagged wild-type STAT3, or the STAT3(C687S), STAT3(C712S) or STAT3(C687S/C712S) mutants, and total cell lysates were heated to the indicated temperatures. Samples were analysed by western blot using anti-Flag antibody. Soluble fractions were plotted versus temperatures. r, HEK293A cells were transfected with Flag-tagged wild-type STAT3, or the STAT3(C687S), STAT3(C712S) or STAT3(C687S/C712S) mutants, and cells were heated to the indicated temperatures. Total cell lysates were analysed by western blot using anti-Flag antibody. Soluble fractions were plotted versus temperatures. In b–i, k, o–r, the experiments were independently repeated at least three times with similar results. For gel source data, see Supplementary Fig. 1.

Source data