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. 2024 Feb 20;7:208. doi: 10.1038/s42003-024-05823-4

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a HeLa cells transiently transfected with FLAG-praja2 (either wild type or ring mutant RM) vector were treated with cycloheximide (10 µM) and harvested at the indicated time points. Total lysates were immunoblotted with anti-FLAG. HSP90 was used as loading control. b Quantitative analysis of the experiments shown in a. A mean value of three independent experiments ± SD is presented. Student’s t-test, *P = 0.02; **P = 0.001. c HeLa cells were serum-deprived overnight and stimulated with EGF (10 ng/ml) in the presence of the protein synthesis inhibitor cycloheximide (10 μM). Where indicated, cells were pre-treated with the proteasome inhibitor MG132 (10 µM). Cells were harvested at the indicated time points and lysed. Lysates were immunoblotted for praja2 and HSP90. d Quantitative analysis of the experiments shown in (c). A mean value of three independent experiments ± SD is shown. Student’s t-test *P = 0.039. e Immunoblot analysis of praja2 and EGFR in HEK293 cells and in two different kidney cancer human cell lines (A-498 and SN12C). Tubulin was used as loading control. f Quantitative analysis of the experiment shown in (e). A mean value of three independent experiments ± SD is shown. Student’s t-test for A-498 *P = 0.02; for SN12C *P = 0.047 (g) A-498 cells were transiently transfected with increasing amount of FLAG-praja2. Cells were harvested 24 h after transfection and lysed. Lysates were immunoblotted with anti-FLAG and anti-EGFR antibodies. h Quantitative analysis of experiment shown in (g). A mean value of three independent experiments ± SD is shown; Student’s t-test for 2ϒ*P = 0.02; Student’s t-test for 4ϒ *P = 0.04. i CaKi-1 cells were transiently transfected with increasing amount of FLAG-praja2. Cells were harvested 24 h after transfection and lysed. Lysates were immunoblotted with anti-FLAG and anti-EGFR antibodies. j Quantitative analysis of experiment shown in (i). A mean value of three independent experiments ± SD is shown.