To verify the changes of gene function and pathways in protein levels, western blot analyses were performed, as described previously [12 (link)]. First, the researchers collected corneal cells from normal individuals and from the family of the KC twins, extracted the total protein using radioimmunoprecipitation assay (RIPA) buffer (Galen, Beijing, China). For each sample, the levels of proteins of interest were normalized to that of GAPDH. Primary antibodies included Collagen IV antibody (ab6586, Abcam), COL15A1 antibody (D162738, Sangon Biotech), COL8A2 antibody (D262652, Sangon Biotech), ACAN antibody (A8536, Abclonal), ADAMTS5 antibody (D260094, Sangon Biotech), ELN antibody (D120588, Sangon Biotech), GPC3 antibody (A1496, Abclonal), MMP3 antibody (ab52915, Abcam), and anti-GAPDH antibody (KC-5G5, Kangchen, Shanghai, China).
Collagen 4 antibody
Manufactured by Abcam
Sourced in China, United States, United Kingdom, Hong Kong
Collagen IV antibody is a laboratory reagent used to detect and quantify the presence of collagen IV, a major structural component of basement membranes, in biological samples. This antibody can be used in various immunoassay techniques to aid in the study of extracellular matrix proteins and their roles in health and disease.
Automatically generated - may contain errors
Lab products found in correlation
Acan antibody, by ABclonal (1 mentions)
Ab52915, by Abcam (1 mentions)
Ab6586, by Abcam (1 mentions)
Cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
α sma antibody, by Abcam (1 mentions)
Sds page, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Ecl system, by Beyotime (1 mentions)
Kdm1 lsd1 antibody, by Abcam (1 mentions)
Sirt3 antibody, by Abcam (1 mentions)
Fibronectin antibody, by Abcam (1 mentions)
Collagen 1 antibody, by Abcam (1 mentions)
Smad3 antibody, by Abcam (1 mentions)
Gapdh, by Beyotime (1 mentions)
Tgf β1, by Proteintech (1 mentions)
Taq pcr mastermix, by Tiangen Biotech (1 mentions)
Cdna synthesis kit, by Takara Bio (1 mentions)
Mmp 9 antibody, by Santa Cruz Biotechnology (1 mentions)
5 protocols using collagen 4 antibody
1
Protein Expression Analysis of Corneal Cells
2
Collagen IV Immunohistochemistry Protocol
3
Immunohistochemical Quantification of Kidney Markers
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For IHC, the specimens were processed under stander protocol and stained with primary antibodies: F4/80 Polyclonal Antibody (Thermo Fisher), Cleaved Caspase-3 antibody (9661, Cell signal, Danvers, MA, USA), and collagen IV Antibody (ab6586, Abcam, Cambridge, MA, USA) overnight at 4 °C. Sections were then incubated sequentially with secondary antibody (goat anti-rabbit or rabbit anti-rat). The positive results were indicated by brown coloration of antigen-containing cells. Finally, Image Pro 6.2 software was used to quantify the positive staining areas of F4/80, Caspase-3, and collagen IV in 10 randomly selected sections of the kidney cortex using ×40 magnification.
4
Protein Extraction and Western Blot Analysis
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Total protein from tissues or cells was extracted by RIPA lysis buffer (Beyotime, China). Equal amount of protein was subjected in 10% SDS-PAGE (Beyotime, China) and then transferred onto PVDF membranes (Millipore, USA). After blocked in 5% skim milk, the membranes were incubated with one of specific primary antibodies overnight at 4 °C: KDM1/LSD1 antibody (1/600; Abcam, UK), α-SMA antibody (1/600; Abcam, UK), Fibronectin antibody (1/600; Abcam, UK), TGF-β1 (1/600; Proteintech, USA), Collagen I antibody (1/600; Abcam, UK), Collagen IV antibody (1/600; Abcam, UK), SIRT3 antibody (1/600; Abcam, UK), Smad3 antibody (1/600; Abcam, UK) and GAPDH (1/8000; Abcam, UK). After incubated with proper second antibody (Beyotime, China) for 1 h at room temperature, the ECL system (Beyotime, China) was used to detect the protein expression and GAPDH was used as control.
5
Investigating Matrix Metalloproteinase Regulation
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Bleomycin was purchased from Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, China). Artesunate was purchased from Guilin Pharmaceutical Co., Ltd. (Guilin, China). Primers were synthesized by Invitrogen Life Technologies (Shanghai, China). The cDNA synthesis kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The total RNA extraction kit and 2x Taq PCR Master mix were obtained from Tiangen Biotech Co., Ltd. (Beijing, China). The TIMP-1 and TIMP-2 antibodies and horseradish peroxidase-conjugated secondary antibodies were purchased from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Collagen-IV antibody was purchased from Abcam Ltd. (Hong Kong, China). MMP-2 antibody and MMP-9 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Hong Kong, China).
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