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. 2018 Jun 21;37(43):5735–5748. doi: 10.1038/s41388-018-0374-1

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© The Author(s) 2018

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 — Jak1-mediated phosphorylation of Stat1 at Tyr701 was responsible for transcription activation of calpain1. a RT-PCR was applied to compare the mRNA levels of calpain1 between p27+/+ and p27−/−(Δ51) cells, and gapdh was used as internal control. b Calpain1 mRNA stabilities were evaluated by real-time PCR in presence of Act D in p27+/+ and p27−/−(Δ51) cells and the results were presented as relative calpain1 mRNA expression. c Mouse calpain1 promoter-driven luciferase reporter was co-transfected together with pRL-TK into p27+/+ and p27p27−/−(Δ51) cells, respectively. Twenty-hour hours post transfection, the transfectants were extracted to evaluate the luciferase activity. TK was used as internal control. The results were presented as relative calpain1 promoter activity, and each bar indicates a mean ± SD from three independent assays. The symbol (*) indicates a significant difference (P < 0.05). d The description of calpain1 promoter with the binding sites of potential transcription factors. e Western blot was used to detect the protein level of Elk-1, JunB, p-Stat1 Y701, Stat1, p-c-Myc S62, and c-Myc in p27+/+ vs p27−/−(Δ51) cells. β-Actin was used as protein loading control. f Western blot was used to determine the expression of JunB, Calpain1, Hsp90 and PHLPP2 in and p27−/−(Δ51/JunB) cells. β-Actin was used as protein loading control. g Mouse calpain1 promoter-driven luciferase reporter was co-transfected together with pRL-TK into p27−/−(Δ51) and p27−/−(Δ51/JunB) cells, respectively. Luciferase activity was evaluated as mentioned above. The results were presented as relative calpain1 promoter activity, and each bar indicates a mean ± SD from three independent assays. The symbol (*) indicates a significant difference (P < 0.05). h Western blot was used to determine the expression of Stat1 Y701F, Calpain1, Hsp90, and PHLPP2 in and p27−/−(Δ51/Stat1 Y701F) cells. β-Actin was used as protein loading control. i Mouse calpain1 promoter-driven luciferase reporter was co-transfected together with pRL-TK into p27−/−(Δ51) and p27−/−(Δ51/Stat1 Y701F) cells, respectively. Luciferase activity was evaluated as mentioned above. The results were presented as relative calpain1 promoter activity, and each bar indicates a mean ± SD from three independent assays. The symbol (*) indicates a significant difference (P < 0.05). j Western blot was used to identify the knockdown efficiency of Jak1 in p27−/−(Δ51) cells, and the protein levels of p-Stat1, Stat1, Calpain1 and PHLPP2 in p27−/−(Δ51) (shJak1) cells. β-Actin was used as protein loading control