Extended Data Fig. 4: LNPK depletion alters eIF2 signaling and translation recovery.
From: Secretome translation shaped by lysosomes and lunapark-marked ER junctions
a, Relative eIF2α levels (normalized to tubulin) in control and LNPK KO U-2 OS cells. Unpaired two-tailed t-test, P = 0.037. b, Relative phosphorylated eIF2α (p-eIF2α) levels in control and LNPK KO cells. Unpaired two-tailed t-test, P = 0.14. c, Immunoblots of ATF4, Tubulin, eIF2α, and p-eIF2α from control (WT), LNPK KO, and U-2 OS cells treated with bortezomib (100 nM, 4 h). All blots imaged under identical conditions. d, Western blots of LNPK, eIF2α, p-eIF2α, ATF4, and Tubulin from control siRNA, LNPK siRNA (LNPK KD), and LNPK siRNA+bortezomib (100 nM, 4 h). e, qPCR quantification of spliced/unspliced XBP-1 ratio in control, LNPK KO, and LNPK KD cells, with or without thapsigargin (1 µM, 1 h). Data show no induction of unfolded protein response by LNPK loss. f, FRAP recovery of cytERM-SUNTAG MS2 puncta in control cells (black, n = 32) vs cycloheximide-treated cells (red, n = 10). Recovery is blocked by cycloheximide, confirming fluorescence recovery reflects new peptide synthesis rather than antibody exchange.
