Western blotting was performed as previously described [32 (link)]. Targeted membranes were incubated with the antibodies for CTHRC1 (Abcam, USA), VEGFA (Abcam, USA) and Actin (Cell Signaling Technology, USA) in 5% milk/tris-buffered saline Tween-20 (TBST) at 4 °C overnight, and then were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling Technology, USA) for 1 h with shaking. After enhanced chemiluminescence (ECL) (Merck Millipore, Germany) reaction, the immunoreactive bands were observed through a Gel Imaging System (Syngene, USA).
Cthrc1
Manufactured by Abcam
Sourced in United States
CTHRC1 is a protein that plays a role in regulating collagen production and the organization of the extracellular matrix. It is involved in various biological processes, including tissue repair and remodeling.
Automatically generated - may contain errors
Lab products found in correlation
Vegfa, by Abcam (1 mentions)
Gel imaging system, by Syngene (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Ab192778, by Abcam (1 mentions)
α tubulin t9026, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Microcon centrifugal filter, by Merck Group (1 mentions)
Ecl plus kit, by Merck Group (1 mentions)
Lamin b1, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Bx51 fluorescence microscope, by Olympus (1 mentions)
Lamp1, by Abcam (1 mentions)
Anti sqstm1, by BD (1 mentions)
Orp150, by Abcam (1 mentions)
Eif2α, by Santa Cruz Biotechnology (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
5 protocols using cthrc1
1
Quantitative Protein Expression Analysis
2
Protein Expression Analysis of Cancer Signaling
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Whole cell lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo, USA) and culture supernatants were concentrated using Microcon centrifugal filters (Millipore, USA). Western blot was performed as previously described [17 (link)]. Primary antibodies against the following proteins were used: CTHRC1 (ab192778, Abcam, USA), p-c-Raf (9427, CST, USA), p-MEK1/2 (9154, CST, USA), p-ERK1/2 (4370, CST, USA), ERK1/2 (4695, CST, USA), p-FRA-1 (5841, CST, USA), FRA-1 (5281, CST, USA), cyclinD1 (2978, CST, USA), snail1 (3879, CST, USA), and MMP14 (13130, CST, USA). α − Tubulin (T9026, Sigma-Aldrich, USA) was used as a loading control.
3
CTHRC1 and β-Catenin Expression Analysis
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Western blotting was performed as previously described [12 (link)]. The antibodies for CTHRC1 (Abcam, Ltd., USA), β-catenin, GAPDH and LaminB1 (Abcam, Cambridge, UK) in 5% milk/TBST (tris-buffered saline Tween-20) were used. The signal was observed by using an enhanced chemiluminescence (ECL) plus kit (Milipore). The relative expression of each protein was determined using NIH Image J software. As for the immunofluorescence assay, cells cultured in the chamber slides were probed with CTHRC1 and β-catenin according to the manufacturer's instructions. The florescein isothiocyanate (FITC)-coupled and Rhodamine (Rhod)-coupled secondary antibodies were purchased from Molecular Probes. Treated cells were observed and pictured through an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan).
4
Western Blot and Immunofluorescence Antibodies
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The following antibodies were used for WB and/or IF as indicated: SIL1 (Proteintech #24110-1-AP); ATF4 (Cell Signaling #11815); ATF6B (Proteintech #15794-1-AP); EIF2α (Santa Cruz #sc-11386); pho-EIF2α (Cell Signaling #3597s); PDI (Assay Design #SPA-891); BiP (BD #610979); ORP150 (Abcam #EP5891); LC3B (Cell Signaling #2775); NCL (Cell Signaling #87792S); NPM (Cell Signaling #3542S); CTHRC1 (Abcam #ab85739); Anti SQSTM1 (BD Bioscience #610833); LAMP1 (Abcam #ab25630); CNX (Cell Signaling #2679S); NES (Millipore #AB5922); CAV-1 (Cell Signaling #3238); and GAPDH (Santa Cruz #sc-32233). Hoechst #33342 from Life Technology was used to counterstain nuclei. For PCR amplification of the spliced and unspliced form of human XBP1, the following primers were used: FW 5′-AGCTCAGACTGCCAGAGATCG-3′; REV 5′-AATCCATGGGGAGATGTTCTA-3′ [35 (link)]. Powders, buffers, and other commonly used reagents were supplied by Sigma unless otherwise noted and were of analytical grade or higher.
5
Immunohistochemical Analysis of CTHRC1 and Integrin β3 in Tumors
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Tumors were collected to fixed and embedded to be made into paraffin sections. Then sections were incubate with primary antibodies against CTHRC1 (Abcam, London, UK) and integrin β3 (Abclonal, Wuhan, China) at 4 °C overnight which were followed by the incubation of the corresponding secondary antibodies and DAB chromogenic reaction and HE staining [38 (link)].
Membranous PSMA quantification was determined by modified H-Score (H-SCORE = ∑ (pi × i) = (percentage of weak intensity area ×1)+(percentage of moderate-intensity area ×2)+(percentage of strong intensity area ×3), to determine the overall percentage of mPSMA positivity across the entire stained tumor sample, yielding a range from 0 to 300 [39 ].
Membranous PSMA quantification was determined by modified H-Score (H-SCORE = ∑ (pi × i) = (percentage of weak intensity area ×1)+(percentage of moderate-intensity area ×2)+(percentage of strong intensity area ×3), to determine the overall percentage of mPSMA positivity across the entire stained tumor sample, yielding a range from 0 to 300 [39 ].
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