Fig. 7

tTmod3-C is accumulated in the nucleus due to its NES sequence loss mediated by AEP cleavage. A Representative images of sub-localization of ZsGreen1-tagged tTmod3-C in A172 and U87-MG cells. Scale bars, 20 μm. B Immunoblots of ZsGreen1-tagged truncations of Tmod3, Lamin B and GAPDH in the indicated A172 and U87-MG cells following sub-cellular fractionation. C, cytoplasm; N, nucleus. C Putative NES and NLS identified in previous work. Conserved amino acids forming the putative NES or NLS are shown in green letters, and the amino acids that were changed by mutagenesis are in purple letters. D A schematic drawing of full-length Tmod3 and its truncations. Summarize their localization in HeLa cells in the absence and presence of Leptomycin B (LMB). E Representative images showing the sub-localization of full-length Tmod3, tTmod3-N and tTmod3-C in HeLa. F Representative images showing the sub-localization of NES or NLS mutant Tmod3 in HeLa cells. G Representative images showing the sub-localization of tTmod3-C with NLS mutation or deletion or NES addition in HeLa cells. H Representative images showing the sub-localization of full-length Tmod3, Tmod3 mutNES, Tmod3 mutNLS and NES-tTmod3-C in HeLa cells treated with LMB. Images are representative of n = 3 independent experiments. Scale bars, 20 μm. I Immunoblots of Tmod3 and GAPDH in the indicated U87-MG and A172cells with ZsGreen1 (empty vector), ZsGreen1-tagged tTmod3-C or its mutants overexpressed. J CCK-8 assay of the indicated U87-MG and A172 cells with ZsGreen1 (empty vector), ZsGreen1-tagged tTmod3-C, ZsGreen1-tagged NES-tTmod3-C or ZsGreen1-tagged tTmod3-C-mutantNLS overexpression. OE = overexpression. ^****P < 0.0001, n.s. = no significance