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. 2022 Aug 1;79(8):461. doi: 10.1007/s00018-022-04489-7

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© The Author(s) 2022

Open AccessThis 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. 2 — CSN-associated USP48 deubiquitinylates nuclear RelA. a AGS cells were transfected with siRNA against USP48 and infected with H. pylori for indicated times. Subcellular fractions were subjected to IB analysis of the indicated proteins. b AGS cells were transfected with siRNA against USP48 and infected with H. pylori for the indicated times (MG132 was added 30 min after infection). IP with an anti-RelA antibody or isotype-matched antibody (IgG) was performed at the indicated times in the presence of NEM and OPT, followed by IB analysis of the indicated proteins. c RelA-IP from the total nuclear fraction of H. pylori-infected AGS cells was incubated with recombinant human USP48 in the presence or absence of NEM, followed by IB analysis of the indicated proteins. d AGS cells were transfected with siRNA against USP48 and infected with H. pylori for the indicated times. Total RNA was isolated at the indicated times and analysed using quantitative RT-PCR for the CXCL8 transcript (the gene of IL-8). Data shown depict the average of triplicate determinations normalized to GAPDH housekeeping gene. Error bars denote mean ± SD. e Equimolar amounts of in-vitro translated Flag-CSN subunits and 0.5 μg of recombinant human USP48 were incubated at 37 °C for 1 h. IP with an anti-USP48 antibody was performed, followed by IB analysis with anti-Flag and anti-USP48 antibodies. f AGS cells were transfected with siRNA against CSN2 and infected with H. pylori for the indicated times (MG132 was added 30 min after infection). IP with an anti-RelA antibody was performed at the indicated times in the presence of NEM and OPT, followed by IB analysis of the indicated proteins. g AGS cells were transfected with siRNA against CSN2 and infected with H. pylori for indicated times. Subcellular fractions were subjected to IB analysis of the indicated proteins. Data information: Data shown in (a–c, e–g) are representative for at least two independent experiments. Data shown in (d) are from three independent experiments with three technical replicates. ***P ≤ 0.001 (Student’s t-test). The band intensities shown in (a, g) were quantified using ImageJ software (NIH). GAPDH, C23 and LaminB2 served as load controls and indicate the purity of the subcellular fractions