anti cathepsin x z p  (R&D Systems)

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: Influence of CTSB and CTSZ on the secretome of tumor cell–macrophage interactions. a Wild-type PyMT cells were co-cultured with macrophages wild type or lacking both Ctsb and Ctsz . After cultivation in serum-free media, the CCM was collected, labeled using dimethylation, fractionated using SCX–HPLC, and subject to LC–MS/MS. b 346 proteins were consistently identified in 3 out of 4 experiments. c Using these proteins, a linear model was fitted and proteins with a change of more/less than 30% and a limma p value ≤ 0.05 were considered to have an altered abundance. The vast majority of proteins show minor quantitative differences. exp experiment, SCX strong cation-exchange chromatography

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques: Cell Culture, Labeling, Liquid Chromatography with Mass Spectroscopy, Chromatography

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: Influence of CTSB and CTSZ on the tumor interstitial fluid composition. a Tumors from PyMT +/0 ; Ctsb +/+ ; Ctsz +/+ and PyMT +/0 ; Ctsb −/− ; Ctsz −/− were carefully dissected and subject to low-speed centrifugation and the TIF was collected. The TIF was depleted of highly abundant proteins and labeled by dimethylation. Samples were either fractionated by SCX (exp1) or by high pH reversed-phase fractionation followed by fraction concatenation (exp2–6) and subject to LC–MS/MS. b Proteins consistently identified in 3 out of 6 experiments and classified as being secreted and/or lysosomal were considered (1139). Only up to three common protein interactions are shown. c A linear model was fitted and proteins with a fold change of more/less than 50% and a limma p value ≤ 0.025 were considered to have an altered abundance. The majority of proteins show minor quantitative differences. exp experiment, SCX strong cation-exchange chromatography

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques: Centrifugation, Labeling, Fractionation, Liquid Chromatography with Mass Spectroscopy, Chromatography

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: Proteins with congruent abundance change due to the absence of Ctsb and Ctsz

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques:

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: CREG1 changes upon Ctsb expression. a CREG1 abundance is increased in both co-cultures having Mϕ Ctsb −/− ; Ctsz −/− , as well as in the TIF of PyMT +/0 ; Ctsb −/− ; Ctsz −/− mice in MS proteomics studies. b Increased abundance is also observed by western blot using the CCM of co-cultures of tumor cell/macrophage deficient of both Ctsb and Ctsz and in co-cultures of wild-type tumor cells with Mϕ Ctsb −/− ; Ctsz −/− . c In the TIF, the abundance of CREG1 changes upon Ctsb expression. CREG1 is increased in the TIF of PyMT +/0 ; Ctsb −/− ; Ctsz +/+ mice, as well as in PyMT +/0 ; Ctsb −/− ; Ctsz −/− mice, but decreased in the TIF with the transgenic expression of human CTSB (Tg(CTSB) +/0 ). CCM cell-conditioned media, TIF tumor interstitial fluid, Mϕ macrophage, wt wild type, dKO deficient from both Ctsb and Ctsz

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques: Expressing, Western Blot, Transgenic Assay

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: CREG1 abundance in breast cancer tissue sections of the MMTV-PyMT model. a Immunostaining of CREG1 in breast tumor sections of PyMT +/0 ; Ctsb −/− ; Ctsz −/− tumors shows stronger staining when compared to wild-type PyMT tumors, especially in stromal cells. A milder effect is seen in tumors from PyMT +/0 ; Ctsb −/− ; Ctsz +/+ mice. No effect was observed in mice deficient for Ctsz . b Lysis of whole tumors shows an important increased abundance of CREG1 in tumors from PyMT +/0 ; Ctsb −/− ; Ctsz +/+ and PyMT +/0 ; Ctsb −/− ; Ctsz −/− , apparent at short exposure (upper lane). After long-exposure CREG1 is observed in tumors from wild-type mice (middle lane). Exp exposure

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques: Immunostaining, Staining, Lysis

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: CREG1 abundance is post-translationally modulated by CTSB inhibition and CTSB induction. a The abundance of CREG1 is increased in PyMT cells treated with the broad spectrum CTSB inhibitor E64d (10 µM). b CREG1 abundance can be reduced upon doxycycline induction (1 µM) of CTSB in a PyMT Ctsb −/− cell line carrying a CTSB doxycycline-inducible vector. c No significant changes of CREG1 at the mRNA level (qRT-PCR) were observed in tumor-cell co-cultures carrying Mϕ lacking both Ctsb and Ctsz compared to wild-type Mϕ (dashed line). d Inhibition of CTSB in PyMT cells with E64d (10 µM) or with CA-074 (10 µM) lead to no significant changes of CREG1 at the mRNA level compared to control (dashed line). n.s. non-significant change, Mϕ macrophages, indCTSB doxycycline-inducible CTSB, DOX doxycycline, CTSB human CTSB

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques: Inhibition, Plasmid Preparation, Quantitative RT-PCR, Control

Journal: Cellular and Molecular Life Sciences

Article Title: The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases

doi: 10.1007/s00018-020-03528-5

Figure Lengend Snippet: CREG1 is partially processed by CTSB generating a neo N-termini. a Partial processing of rCREG1 (2 µg) is observed by incubation with rCTSB (200 ng) after 6 h and increased after 24 h at pH 5.0. Incubation of rCREG1 with rCTSZ does not have significant changes; whereas, incubation of rCREG1 with both rCTSB and rCTSZ leads to a moderate increase in the processing at 6 and 24 h. At pH 6.6, no changes are observed with neither rCTSB, rCTSZ, nor with both CTSB and CTSZ. b N-terminal sequencing (Edman degradation) done with samples treated or not with rCTSB or rCTSB and rCTSZ after 24 h identified a potential CTSB cleavage site (Arrow H 37 –G 38 bond). The sequence R 32 GGRD (blue) was identified in non-treated rCREG1 and the same neo N-termini GDWDV (red) was identified in samples treated with rCTSB or rCTSB and rCTSZ. Signal peptide (dark yellow); rCREG1 recombinant murine CREG1, rCTSB recombinant murine CTSB, rCTSZ recombinant murine CTSZ

Article Snippet: After blocking of membranes with 4% non-fat milk in PBS-Tween, they were incubated with primary antibodies goat anti-mouse CREG1 (R&D systems, Minneapolis, MN, USA; AF1697), goat anti-mouse CTSB (R&D systems; BAF965), goat anti-human CTSB (R&D systems; AF953), goat anti-mouse CTSZ (R&D systems; BAF1033), or mouse anti-alpha-tubulin (Sigma-Aldrich; T9026) overnight at 4 °C.

Techniques: Incubation, Sequencing, Recombinant