Fig. 5: APT2 mediates the depalmitoylation of GPX4.
From: Palmitoylation-dependent regulation of GPX4 suppresses ferroptosis
a Analysis of proteins interacting with GPX4 in A375 and 293T cells infected with lentiviruses encoding empty vector or Flag-GPX4, using anti-Flag magnetic beads enrichment and LC-MS/MS. b Co-immunoprecipitation in 293T cells transiently transfected with Flag-GPX4 and HA-APT2 plasmids to investigate GPX4-APT2 interaction detected by immunoblots. c Immunofluorescence staining for GPX4 and APT2 was performed in A375 cells infected with lentiviruses encoding a control plasmid or an HA-APT2 plasmid. Scale bar, 4 μm. d Palmitoylation analysis of exogenous GPX4 in A375 cells infected with lentiviruses encoding control or APT2 shRNA, using the ABE assay with or without HAM. e Immunoblots of APT2, GPX4, SLC7A11, and ACSL4 in A375 and HT1080 cells infected with lentiviruses encoding control or APT2 shRNA. f RT-qPCR detection of GPX4 mRNA levels in A375 and HT1080 cells infected with lentiviruses encoding control or APT2 shRNA. g Immunoblots of GPX4, FSP1, SLC7A11, and ACSL4 in A375, HT1080, and A549 cells treated with ML349 at indicated concentrations. h RT-qPCR analysis of GPX4 mRNA levels in A375 cells treated with ML349 at indicated concentrations. i, j Immunoblots of GPX4 and APT2 in A375 cells infected with lentiviruses encoding control or APT2 shRNA, treated with CHX for indicated times (i). Quantification of GPX4 protein levels (j). Data are presented as mean ± SD. Statistical analysis: two-way ANOVA with Tukey’s test for (j); one-way ANOVA with Tukey’s test for (f, h). Sample sizes: (f, h, j) n = 3 independent experiments. Image in b, c, d, e, g, i shows representative results from three independent experiments.
