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. 2023 May 24;14(5):341. doi: 10.1038/s41419-023-05862-5

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© The Author(s) 2023

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 — A Silver staining of biotinylated PAXIP1-AS1-associated proteins. B Western blotting of proteins from PAXIP1-AS1 and its antisense pull-down assays; n = 3. C The association of endogenous PABPC1 and PAXIP1-AS1 in MKN-74 cells was detected by RNA immunoprecipitation (RIP) assay using anti-PABPC1 antibodies. Relative enrichment (mean ± SD) of PAXIP1-AS1 with anti-PABPC1 or anti-IgG was measured by qPCR. D, E Western blotting of PABPC1 in samples pulled down by full-length or truncated PAXIP1-AS1 (F1: 1–600, F3: 601–1200, F3: 1201–1800, F4: 1801–2275); n = 3. F, G Full-length and truncated PABPC1 plasmids with 6× His-tag were transfected into MKN-74 cells for 48 h and extracted for RIP assays with anti-His antibody. Results showed that PAXIP1-AS1 mainly bound to the RRM4 region and PABC domain in full-length PABPC1. H Western blotting analysis of PABPC1 in Control and PAXIP1-AS1-overexpressed GC cells. I Total RNA from Control or PAXIP1-AS1-overexpressing AGS and MKN-74 cells was analysed by qPCR to validate PAXIP1-AS1 expression (the left panel) and determine PABPC1 mRNA levels (the right panel). ****P < 0.001 and *P > 0.05, Control vs. PAXIP1-AS1. n = 3. J Control or PAXIP1-AS1-overexpressed MKN-74 cells were treated with 100 μM CHX for indicated periods. Subsequently, cell lysates were analysed by western blotting with anti-PABPC1 and anti-GAPDH antibodies. K PAXIP1-AS1 or control plasmid was transfected into MKN-74 cells for 48 h and cells were treated with DMSO, CHX (100 μM), or CHX plus MG132 (10 nM) for 0, 6, or 12 h before protein extraction. Subsequently, cell lysates were analysed by western blotting with anti-PABPC1 and anti-GAPDH antibodies. L PAXIP1-AS1-overexpressed or control plasmid was transfected into MKN-74 cells for 48 h and cells were treated with 10 nM MG132 for 6 h. Extracts were immunoprecipitated with anti-PABPC1 antibody, and PABPC1 polyubiquitination was examined by western blotting using anti-ubiquitin antibody.