Cathepsin X/Z/P Antibody

Detection of Cathepsin X/Z/P by Immunocytochemistry/ Immunofluorescence

The effects of simultaneous TLR3/4 activation on cathepsin X expression and activity in microglia. A–D BV2 cells or primary microglia cultures were stimulated with 1 µg/mL LPS and/or 10 µg/mL poly(I:C) in serum-free medium for the times indicated (A, C) or for 24 h (B, D). A, B Cathepsin X protein levels in cell lysates of the control and stimulated cells were measured by ELISA, using a goat anti-cathepsin X capture antibody (AF934) and a mouse anti-cathepsin X detection antibody (3B10). Data are presented as means ± standard deviation of at least two independent experiments, each performed in duplicate. C, D Cathepsin X activity in cell lysates was determined using the cathepsin X-specific substrate Abz-Phe-Glu-Lys(Dnp)-OH. Data are presented as means ± standard deviation of at least two independent experiments, each performed in duplicate. *p ˂ 0.05. E Representative images of double immunofluorescence staining of cadherin (green) and cathepsin X (red) in unstimulated BV2 cells (control) and in BV2 cells stimulated with LPS and/or poly(I:C) for 24 h. Nuclei were counterstained with DAPI (blue). Right: quantification of the relative co-localization areas of cadherin and cathepsin X, presented as means ± standard deviation of pixels (cell numbers ≥ 10). Scale bars: 10 µm. *p ˂ 0.05 Image collected and cropped by CiteAb from the following open publication (https://link.springer.com/10.1007/s12035-021-02694-2), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Cathepsin X/Z/P by Western Blot

Analysis of the endogenous expression and activity of cathepsins in HeLa and SH-SY5Y cells. A Previously published [75] RNA sequencing data from HeLa and SH-SY5Y cells, publicly available through the Human Proteome Atlas portal, show transcripts per million (TPM) for 10 human cathepsins. B Immunoblot and protein expression analysis of endogenous CTSB, CTSD, CTSL and CTSZ expression in HeLa and SH-SY5Y wildtype (WT) cells. The signal intensities of all detected bands with respective CTS-antibodies were normalized to GAPDH. C The quenched cysteine-protease activity-based probe (ABP) BMV-109 contains a fluorophore-quencher pair. Once the ABP binds covalently to the active-site cysteine, the quenching group is released, and the protease is irreversibly labelled, resulting in the emission of fluorescence that can be detected via fluorescent SDS-PAGE. The in-gel fluorescence emission of the scanned SDS-PAGE shows BMV-109-labelled HeLa and SH-SY5Y WT cells treated with or without 50 μM E64D (cysteine-protease inhibitor). Cy5 signal was detected at excitation at 635 nm. Total protein staining with Coomassie Brilliant Blue was used as a loading control. D The immunoblot shows a successful generation of two HeLa CTSB, CTSD, CTSL, CTSZ, and CTSBDLZ KO clones (D) and of SH-SY5Y CTSB, CTSD, CTSL and CTSBDL clones (E) each. F Fluorescent SDS-PAGE of BMV-109-labelled HeLa (F) and SH-SY5Y (G) WT, single KO, and multiple CTS-deficient cells. kDa kilodalton, * indicates non-specific-binding of the antibody, p pro, m mature. sc-single chain, dc-double chain (A, C) “Created with BioRender.com.” Image collected and cropped by CiteAb from the following open publication (https://link.springer.com/10.1007/s00018-024-05274-4), licensed under a CC-BY license. Not internally tested by R&D Systems.