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. 2021 Mar 19;40(16):2858–2871. doi: 10.1038/s41388-021-01729-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Presence of AKT in immunoprecipitate of TMCC3 from AS-B634 using anti-TMCC3 antibody. b, c MDA-MB231 cells transfected with flag-tagged full-length (FL)-TMCC3 and HA-tagged AKT were subject to co-immunoprecipitation assay using anti-flag antibody (b) or anti-HA antibody (c), followed by immunoblotting. d Diagram of full-length TMCC3 and truncated forms of TMCC3 containing 1-153 a.a., 1-282 a.a., 154-282 a.a., 283-416 a.a. e Co-immunoprecipitation analysis of flag-tagged TMCC3 with HA-tagged AKT proteins. 293T cells were transfected with a plasmid encoding HA-tagged AKT and another plasmid encoding flag tag alone or various flag-tagged TMCC3 forms: full-length (FL), 1-153 a.a., 1-282 a.a., 154-282 a.a., or 283-416 a.a. f Flowchart of luminex immunosandwich assay. Detailed procedures were described in “Materials and methods”. g Mean fluorescence intensities (MFI) showed AKT bound to beads coupled with 1-158 a.a. domain of TMCC3 proteins (n = 4). h Levels of pAKT^S473 and total AKT were examined in flag-tagged full-length (Flag-FL), 1-153 a.a.- or 154-477 a.a.-truncated TMCC3 transfected MCF7 and MDA-MB231 (MB231) by western blot analysis. Mammosphere forming capacities (i) and migration abilities (j) were assessed in cells transfected with flag-tagged full-length (Flag-FL), 1-153 a.a.- or 154-477 a.a.-truncated TMCC3. i Sphere numbers were calculated after culture for 7 days. ^#p < 0.001, ^##p < 0.0001 (n = 8, t-test). j The numbers of migrated cells in each group were determined in trans-well migration assay. *p < 0.05, **p < 0.01 (n = 3, t-test).