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. 2021 Aug 10;12:4826. doi: 10.1038/s41467-021-25157-7

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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. 4 — a, bNek1^+/+ and Nek1^Kat2J/Kat2J MEFs were transfected with shRNA targeting Vps26b or scrambled shRNA for 7 days. The cells were then pretreated with 10 μM Nec-1s for 30 min, followed by treatment with 1 ng/mL TNF + TAK1 inhibitor 500 nM 5z 7-oxozeaenol (5z7) (a) or 10 ng/mLTNF + 50 nM SM164 + 20 µM zVAD.fmk (b) for 5 h. The cell death was measured with ToxiLight. Mean ± SD. n = 3 biological repeats. Two-way ANOVA with Dunnett’s test. c Volcano plot analysis of mass spectrometry data comparing the plasma membrane proteins of Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs enriched by Pierce™ Cell Surface Protein Isolation Kit. n = 3 biological independent repeats. Unpaired two-tailed Student’s t test. d Heatmap analysis of known retromer substrates that showed statistically significant difference in the plasma membrane presence between Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs in the mass spectrometry analysis (c). e Nek1^+/+ and Nek1^Kat2J/Kat2J MEFs were lysed with NP-40 buffer, and cell lysates were immunoprecipitated with anti-VPS26B antibody. Whole cell lysates and immunoprecipitated proteins were analyzed by western blotting with indicated antibodies. Arrowhead points to the molecular weight expected for VPS26B. Uncropped blots in the Source Data file. f NEK1 phosphorylates VPS26B in a Ser302/304 dependent manner. 293 T cells were transfected with expression vectors for HA tagged NEK1 and FLAG tagged VPS26B^WT, or VPS26B^S302A/S304A for 24 h. The cells were then lysed with NP-40 buffer and immunoprecipitated with anti-HA or anti-FLAG antibody conjugated agarose. Purified FLAG-tagged VPS26B^WT and VPS26B^S302A/S304A were incubated with purified HA-NEK1 (30 °C. 1 h) in kinase reaction buffer. The reaction products were analyzed by western blotting with indicated antibodies. Uncropped blots in the Source Data file. g–i 293 T cells were transfected with expression vectors for FLAG-tagged VPS26B^WT, VPS26B^S311D, VPS26B^S311A, VPS26B^S302D/S304D or VPS26B^S302A/S304A for 24 h and then lysed with NP-40 buffer. The cell lysates were immunoprecipitated with anti-FLAG antibody conjugated agarose. The whole-cell lysates and immunoprecipitated proteins were analyzed by western blotting with indicated antibodies (g). Uncropped blots in the Source Data file. SNX27 (h) and SNX2 (i) binding to VPS26B was normalized to immunoprecipitated FLAG-VPS26B and quantified using ImageJ. n = 3 repeated experiments and blots. Data are presented as mean ± SD. One-way ANOVA with Dunnett’s test.