Western blot analysis was conducted using a previously described protocol.39 (link) We used antibodies against ANXA1, β-catenin, caspase 3, Lrp5, Lrp6, Runx2, Sclerostin, Snail, TGFβ, NFATc1, cathepsin K (all from Cell Signaling, Danvers, MA, USA), DMP1, ANXA6, CXCL5 (all from Abcam, Cambridge, MA, USA), M-CSF, MMP9, OPN, TPM4 (all from Santa Cruz, Dallas, TX, USA), WISP1 (R&D systems, Minneapolis, MN, USA), β-actin (Sigma, Saint Louis, MO, USA), LIMA1, Trail (both from Novus, Centennial, CO, USA), p53, CXCL1 (both from Invitrogen, Carlsbad, California, USA), and DSP (ProteinTech, Rosemont, IL, USA). The expression levels of Sclerostin and Lrp5 in CM were detected by ELISA (My BioSource, San Diego, CA, USA). Proteins isolated from A5 osteocyte CM, Y4 osteocyte CM, and osteoclast control CM (RAW264.7 cells) were analyzed with an HF Hybrid Quadrupole Orbitrap mass spectrometer. Among the 549 identified proteins, 49 proteins had higher expression levels in A5 CM than in Y4 CM and control CM. Among these proteins, 11 (p53; SPARC = osteonectin; TPM1, TPM4 = tropomyosin 1 and 4; ANXA1, ANXA6 = annexin A1 and A6; FMOD = fibromodulin; OGN = osteoglycin; DSP = desmoplakin; AHNAK = desmoyokin; and LIMA1 = LIM domain actin-binding protein 1) were identified as potential tumor suppressors.
Cxcl5
Manufactured by Abcam
Sourced in United States, United Kingdom
CXCL5 is a chemokine that plays a role in the immune response. It is involved in the recruitment and activation of neutrophils. CXCL5 is expressed in various cell types, including epithelial cells, endothelial cells, and immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Abcam (3 mentions)
Hrp conjugated anti rabbit antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (2 mentions)
E cadherin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
Ecl enhanced chemiluminescence detection kit, by GE Healthcare (2 mentions)
Cxcl1, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Cell Signaling Technology (1 mentions)
Cathepsin k, by Cell Signaling Technology (1 mentions)
Sclerostin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Mmp 9, by Santa Cruz Biotechnology (1 mentions)
Dmp 1, by Abcam (1 mentions)
Anxa1, by Cell Signaling Technology (1 mentions)
Anxa6, by Abcam (1 mentions)
M csf, by Santa Cruz Biotechnology (1 mentions)
Wisp1, by R&D Systems (1 mentions)
8 protocols using cxcl5
1
Proteomic Analysis of Osteocyte Secretome
2
Cytokine Quantification by ELISA
3
Quantifying Key Proteins in Proteomics
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The key proteins, where clear differences were found during the proteome profiler measurements, were quantified using custom Human Magnetic Luminex Assay (CHI3L1, C5a, EGF, CD40L, VEGF, CRP, Il-17a, Osteopontin, Angiopoetin, IL-1RA, EMMPRIN, Lipocalin-2, Pentraxin-3, PDGF-AA, PDGF-BB) (R&D Systems Inc.) or ELISA assays (MPO, ALCAM, CD97, C1qR1, TGF-beta, Fibrinogen, Thrombospondin-1, CXCL-5) (Abcam, Cambridge, UK) (n = 8)
4
Western Blot Analysis of EMT Markers
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Equal amounts of whole-cell extracts were resolved by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% nonfat dry milk in TBST and incubated (2 h) with the indicated primary antibodies (CXCL5, E-cad, N-cad, Vimentin, CD44, P-AKT, T-AKT, P-NFκB and T- NFκB; 1:1000 dilution), followed by incubation (1 h) with goat anti-rabbit or anti-mouse secondary antibodies (1:5000 dilution; Sigma-Aldrich). Immunoreactivity was visualized by an enhanced chemiluminescence reagent (Perkin Elmer Cetus, Foster City, California, USA). To demonstrate equal loading, the blots were stripped and reprobed with a specific antibody against GAPDH (1:5000 dilution; Sigma-Aldrich). The antibodies against CXCL5, E-cad, N-cad, Vimentin, CD44, P-AKT, T-AKT, P-NFκB and T- NFκB were purchased from Abcam Biotechnology (Abcam, Cambridge, UK). All the measurements were performed in triplicate.
5
Western Blot Analysis of EMT Markers
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The total proteins were lysed by RIPA Lysis Buffer (Sigma, USA) containing 10% PMSF (Sigma). SDS-PAGE was applied to separate the protein, and then, the blots were electrotransferred to PVDF membranes (Millipore, USA). After being blocked by 5% fat-free milk at room temperature for 1 h, the membranes were incubated with primary antibodies, such as CXCL5 (1 : 1000; Abcam, Cambridge, USA), E-cadherin (1 : 1000; Abcam), N-cadherin (1 : 1000; Abcam), Vimentin (1 : 1000; Abcam), p-P65 (1 : 1000, Abcam), and P65 (1 : 1000, Abcam). Next, the blots were incubated by a secondary anti-rabbit HRP-conjugated antibody (Cell Signaling). The protein signals were captured using an Enhanced Chemiluminescence Detection Kit (ECL, Pharmacia Biotech, Arlington, USA).
6
Western Blot Analysis of sEV Proteins
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Proteins from sEVs and cells were extracted using RIPA solution plus protease inhibitors. We measured protein concentration with a BCA protein assay kit (Solarbio Life Science, Beijing, China). The SDS-polyacrylamide gel electrophoresis was used for protein separation and then was transferred onto polyvinylidene difluoride membranes. Five percent skim milk was used to block the membranes before primary antibodies were added.
The primary antibodies included CD9 (1:2000; Abcam, Cambridge, MA, USA), CD63 (1:1000; Abcam), TSG101 (1:1000; Proteintech, Chicago, IL, USA), CXCL5 (1:2000; Abcam), and β-actin (1:5000; Cell Signaling Technology, Danvers, MA, USA). After three 10-minute washes with Tris-buffered saline with Tween 20, incubation with secondary antibodies (1:5000; Cell Signaling Technology) followed. The protein bands were detected by enhanced chemiluminescence and quantified by ImageJ software.
The primary antibodies included CD9 (1:2000; Abcam, Cambridge, MA, USA), CD63 (1:1000; Abcam), TSG101 (1:1000; Proteintech, Chicago, IL, USA), CXCL5 (1:2000; Abcam), and β-actin (1:5000; Cell Signaling Technology, Danvers, MA, USA). After three 10-minute washes with Tris-buffered saline with Tween 20, incubation with secondary antibodies (1:5000; Cell Signaling Technology) followed. The protein bands were detected by enhanced chemiluminescence and quantified by ImageJ software.
7
Western Blot Analysis of Protein Expression
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The total proteins were lysed by RIPA Lysis Buffer (Sigma, USA) containing 10% PMSF (Sigma). The SDS-PAGE was applied to separate the protein and then the blots were electro-transferred to PVDF membranes (Millipore, USA). After being blocked by 5% fat-free milk at ream temperature for 1 h, the membranes were incubated with primary antibodies. The primary antibodies were against CXCL5 (1:1000; Abcam, Cambridge, USA), E-cadherin (1:1000; Abcam), N-cadherin (1:1000; Abcam), Vimentin (1:1000; Abcam), c-Myc (1:1000, Abcam ), TRAF6 (1:1000, Abcam). Next, the blots were incubated by secondary anti-rabbit HRP-conju gated antibody (Cell Signaling). The protein signals were captured using Enhanced Chemiluminescence Detection Kit (ECL, Pharmacia Biotech, Arlington, USA).
8
Quantitative Analysis of ONZIN Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, 1 µg of total RNA was used to generate cDNA with the FastQuant RT Kit (Tiangen Biotech, Beijing, China). Quantitative PCR primers for human ONZIN included a forward primer (5'-TTCAGCGACTGCGGAGTCT-3') and reverse primer (5'-CCCAAGGCACGGGAAA-3'), while β-actin was used as an internal control (forward primer, 5'-AGAGGGAAATCGTGCGTGAC-3'; reverse primer, 5'-CAATAGTGATGACCTGGCCGT-3').
Cell lysates were separated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gel and immunoblotted overnight at 4°C with antibodies against ONZIN, CXCL5 (Abcam, Cambridge, MA, USA), vinculin, Flag M2 (Sigma-Aldrich, St. Louis, MO, USA), p-AKT, AKT, ERK, and p-ERK (Cell Signaling Technology, Beverly, MA, USA). The signals were detected using a chemiluminescence phototype-HRP kit (Bio-RAD, Hercules, California, USA).
Cell lysates were separated by 10% or 15% sodium dodecyl sulfate-polyacrylamide gel and immunoblotted overnight at 4°C with antibodies against ONZIN, CXCL5 (Abcam, Cambridge, MA, USA), vinculin, Flag M2 (Sigma-Aldrich, St. Louis, MO, USA), p-AKT, AKT, ERK, and p-ERK (Cell Signaling Technology, Beverly, MA, USA). The signals were detected using a chemiluminescence phototype-HRP kit (Bio-RAD, Hercules, California, USA).
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