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. 2022 Apr 26;10:37. doi: 10.1038/s41413-022-00200-5

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© The Author(s) 2022

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. 4 — Smad 1 degradation is less efficient in Flnb^−/− -transfected MEFs due to FLNB interaction with i-Smads. a Western blots of Flnb^+/+ and Flnb^–/– MEF lysates nontransfected or transfected with Smad 1-Flag plasmid, ubiquitin-HA plasmid, or both. Blots were probed using antibodies against Flag, HA, and Actin. b Quantification of total Smad 1-Flag levels in Flnb^+/+ versus Flnb^–/– MEFs normalized to actin levels. The results show that more Smad 1-Flag plasmid remained after transfection and that the 24-h recovery period indicated inefficient degradation in the absence of FLNB. c Quantification of total Smad 1-Flag levels in Flnb^+/+ versus Flnb^–/– MEFs transfected with ubiquitin normalized to both Actin levels and Smad 1-Flag levels without ubiquitin transfection. The results again show that more Smad 1-Flag plasmid remained after ubiquitin transfection, indicating a lower efficiency in ubiquitinated degradation in the absence of FLNB. n = 3, *P < 0.05, ***P < 0.001. d Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 1-Flag plasmids immunoprecipitated using an antibody against GFP. FLNB western blot was probed using a GFP antibody, and Smad 1 western blot was probed using a Flag antibody. The results showed the detection of Smad 1-Flag protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 1. e Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 6-HA plasmids immunoprecipitated using an antibody against GFP. The results showed the detection of Smad 6-HA protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 6. f Western blots of lysates isolated from wild-type MEFs transfected with FLNB-GFP and Smad 7-HA plasmids immunoprecipitated using an antibody against GFP. The results showed the detection of Smad 7-HA protein when lysates were immunoprecipitated using a GFP antibody, confirming the in vitro interaction of FLNB and Smad 7. n = 3, WCL Whole Cell Lysate, IP Immunoprecipitate