mouse anti cstb  (Santa Cruz Biotechnology)

Journal: Cells

Article Title: Stefin B and Cystatin C Deficiency Suppresses Tumor Growth and Alters Tumor Microenvironment in a Breast Cancer Model

doi: 10.3390/cells15040360

Figure Lengend Snippet: Expression of stefin B and cystatin C, and cysteine cathepsin activity, in PyMT mammary tumors of different genotypes. ( A ) Western blot for stefin B (StfB) in mammary tumors from wild-type (PyMT;WT; n = 3), stefin B knockout (PyMT; StfB −/− ; n = 3), cystatin C knockout (PyMT; CstC −/− ; n = 3), and double-knockout (PyMT;DKO; n = 3) mice at 14 weeks of age. StfB is absent in PyMT; StfB −/− and PyMT;DKO tumors, confirming successful knockout. β-actin was used as a loading control. ( B ) Western blot for cystatin C (CstC) in the same tumor samples. CstC is absent in PyMT; CstC −/− and PyMT;DKO tumors, verifying successful knockout. β-actin was used as a loading control. ( C ) Relative cathepsin activity determined in homogenates of PyMT tumors (PyMT;WT, n = 4; PyMT;DKO, n = 3). Data represent biological replicates from independent tumors derived from different mice. Activity was measured as Z-Phe-Arg-AMC hydrolysis in the presence or absence of E64. Differences were analyzed using Student’s t -test. ( D ) Relative cathepsin activity determined in cell culture medium of primary tumor cells (PyMT;WT, n = 3; PyMT;DKO, n = 3). Data represent biological replicates from independent primary tumor cell isolates derived from different mice. Activity was measured as Z-Phe-Arg-AMC hydrolysis in the presence or absence of E64. Differences between groups were analyzed using Student’s t -test.

Article Snippet: Membranes were probed with anti-CstC antibody (Abcam, Cambridge, UK; ab109508, 1:100,000) and anti-StfB antibody (R&D Systems, Minneapolis, MN, USA; MAB1409, 1:5000).

Techniques: Expressing, Activity Assay, Western Blot, Knock-Out, Double Knockout, Control, Derivative Assay, Cell Culture

Journal: Biomedical Reports

Article Title: Identification of diagnostic upper gastrointestinal cancer tissue type-specific urinary biomarkers

doi: 10.3892/br.2019.1190

Figure Lengend Snippet: Validation of identified proteins by western blotting. Urine samples were separated by 20% SDS-PAGE and analysed by western blotting using antibodies specific against CSTB, CTSB and albumin. Panels (A) and (C) (left) show the western blotting results of combined urine samples in the 1-100-kDa range. The samples were tested initially for the presence of fragments of CSTB and CTSB in the region of the measured m/z (open triangles), as well as full-length molecules (filled triangles) or other breakdown products. Reliable signals in the 35-kDa range for CSTB and 25-kDa for CTSB were then further analysed. Validation and confirmation of LC-MS/MS and Mascot results are shown in the strip-blots in panels (A) and (C), which show the results of (A) 8 or (C) 7 urine samples that were used in LC-MS/MS and subsequent Mascot searches, together with the cluster peak intensity matrices derived from the SELDI analysis (underneath the individual blots). Panels (B) and (D) depict the analysis of four random cancer and four control samples. The samples were selected based on the IMAC30 SELDI analysis for the presence of a peak cluster at m/z 2577 for CSTB, and on the CM10 SELDI peak pattern at m/z 2447 for CTSB (cancer samples) or the absence of these m/z peaks (healthy controls). ‘e’ corresponds to an estimated value (in brackets), where no peak at the m/z point could be detected above the S/N ratio. Qualitative loading control was performed by probing against the common urinary molecule serum albumin. Molecular weight indicators are depicted on the left, and the target proteins on the right of the blots. The cancer locus is the histologically assessed cancer site of duodenum (D), pancreas (P), oesophagogstric junction (OGJ), oesophagus (O), or non-cancerous control (C). CTSB, cathepsin-B; CSTB, cystatin-B; SELDI, surface-enhanced laser desorption/ionization; MS, mass spectrometry; S/N, signal-to-noise ratio.

Article Snippet: The antibodies used were rabbit anti-human CTSB (G-60; 1:1,000; cat. no. 3373; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-human serum albumin (1:1,000; cat. no. A3293; Sigma-Aldrich; Merck KGaA), mouse anti-human CSTB (1:400; cat. no. sc-101510; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and the peroxidase-coupled secondary antibodies were from Upstate (Lake Placid, NY, USA), used at a dilution of 1:5,000.

Techniques: Biomarker Discovery, Western Blot, SDS Page, Liquid Chromatography with Mass Spectroscopy, Stripping Membranes, Derivative Assay, Control, Molecular Weight, Mass Spectrometry