Human Cathepsin S Antibody

Cathepsin S in Human Lymph Node.

Cathepsin S was detected in immersion fixed paraffin-embedded sections of human lymph node using Goat Anti-Human Cathepsin S Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1183) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of immersion fixed paraffin-embedded Tissue Sections.

Detection of Human Cathepsin S by Western Blot

CTSS attenuates EGF-mediated EGFR degradation.(a) OEC-M1 and MDA-MB-231 cells were pretreated with 20 μM 6r or ZFL for 1 h and subsequently incubated with 100 ng/mL EGF for an additional 2 h. The total cell lysates were analysed using EGFR-specific antibodies. ACTIN was used as the internal control for semiquantitative loading in each lane. (b) The cells were stimulated with EGF (100 ng/mL) with or without the pretreatment of 20 μM 6r for the indicated durations. EGFR degradation was examined through immunostaining by using an anti-EGFR antibody. Notably, a substantial amount of EGFR was detectable even after 6 h of EGF stimulation in 6r-treated cells. (c) The OEC-M1 cells were transiently transfected with plasmids (pCMV) that encoded wild-type CTSS. After 24 h of transfection, the cells were treated with 100 ng/mL EGF for the indicated durations and the cellular EGFR, CTSS, and ACTIN signals were determined through Western blotting. The lifespan of EGF-mediated EGFR degradation was calculated by normalising the signal intensity of EGFR with that of ACTIN. (d) The MDA-MB-231 cells were transfected with specific 50 nM CTSS siRNA (si-CTSS) for 24 h and subsequently incubated with 100 ng/mL EGF for the indicated durations. The nontargeting scramble siRNA (si-SC) was used as the scramble control. (e) The MDA-MB-231 cells were transiently transfected with plasmids encoding the CTSS-C25A mutant. After 24 h of transfection, the cells were incubated with 100 ng/mL of EGF for the indicated durations. Furthermore, the cells were harvested and subjected to SDS-PAGE and Western blotting. EGFR degradation was determined using an antibody against EGFR. ACTIN signalling was included as the loading control. Image collected and cropped by CiteAb from the following open publication (https://www.nature.com/articles/srep29256), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Cathepsin S by Western Blot

CTSS inhibition does not impair lysosomal activities.(a) OEC-M1 cells were pretreated with vesicle or 100 nM BAF for 1 h and then incubated with 20 μM 6r for 1 h further. Lysosomal proteolytic activities were determined using BODIPY–BSA and quantified through flow cytometry. (b) After 48 h of siRNA knockdown of CTSS and CTSB, the relative expression of CTSS and CTSB were determined through Western blotting (right panel). Lysosomal proteolytic activities were determined using BODIPY–BSA and quantified through flow cytometry (right panel). Data represent the mean ± SD of three independent experiments. Differences were found to be statistically significant at ***P < 0.001. Image collected and cropped by CiteAb from the following open publication (https://www.nature.com/articles/srep29256), licensed under a CC-BY license. Not internally tested by R&D Systems.