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. 2022 Jun 30;13(6):574. doi: 10.1038/s41419-022-05024-z

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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. 5 — A Immunohistochemical staining images of CA-IX expression in control brain and GBM specimen (Scale bar-100 μm). B The mRNA obtained for Gal-1 and CA-IX from TCGA datasets showed a positive correlation. C Western blot analysis of CA-IX in shGal-1 stable and SV treated GSC20 and GSC33 cells. The density levels were quantified and represented as a bar graph. D MTT assay. E Expression of CA-IX and Gal-1 in untreated controls and samples treated with different concentrations of CA-IX inhibitor, SLC-0111, in GSC20 and GSC33 cells. F Coomassie Blue-stained SDS-PAGE gel showing the expression levels of GST-Gal-1 protein after induction with 0.5 mM IPTG for 3 h. G Co-immunoprecipitation (GST pulldown) assay was performed with Gal-1 and CA-IX antibodies, using GST-tagged Gal-1 fusion purified protein lysate as prey and GSC20/GSC33 control total protein as bait. Eluted proteins were resolved by electrophoresis and subjected to immunoblot analysis. GST is shown as a loading control. H PLA of Gal-1 and CA-IX were performed in GSC20 and GSC33 cells. The representative micrograph of Gal-1 + CA-IX shows the positive PLA. Red spots confirmed the association. Single antibody stain with Gal-1 and CA-IX showed negative results. DAPI was used to stain the nuclei.