The proteins were extracted from cells or tissue, and Western blotting was performed as previously described.[21 (link)] Equal amounts of proteins were separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked in 5% nonfat dry milk for 1 h, followed by incubation with primary antibodies against FSTL1 (1:500 dilution, Santa Cruz Biotechnology, CA, USA), Collagen I (1:200 dilution, Abcam, Cambridge, UK), β-tubulin (1:1,000 dilution, Abcam, Cambridge, UK), phosphorylated (p)-p38, p38, p-ERK, ERK, p-JNK, and JNK (1:200 dilution, Cell Signaling Technology, MA, USA), α-SMA (1:500 dilution, Sigma-Aldrich, St. Louis, MO, USA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:3,000 dilution, Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C. The protein bands were visualized using the LI-COR Odyssey infrared double-fluorescence imaging system (LI-COR, Lincoln, NE, USA). The value of the relative density of each target protein band was normalized to the density of the corresponding β-tubulin or GAPDH band.
Odyssey infrared double fluorescence imaging system
Manufactured by LI COR
Sourced in United States
The LI-COR Odyssey infrared double-fluorescence imaging system is a laboratory instrument designed for the simultaneous detection and analysis of two fluorescent signals. It utilizes near-infrared fluorescence technology to capture high-resolution images of protein and nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
Irdyetm700 red conjugated affinity purified anti rabbit or anti mouse igg, by LI COR (2 mentions)
α sma, by Merck Group (1 mentions)
P jnk and jnk, by Cell Signaling Technology (1 mentions)
Collagen 1, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
β tubulin, by Abcam (1 mentions)
Fstl1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Anti cyto c, by Abcam (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ab32199, by Abcam (1 mentions)
Ab99421, by Abcam (1 mentions)
Mini protean electrophoresis module assembly, by Bio-Rad (1 mentions)
Mini trans blot electrophoresis transfer cell, by Bio-Rad (1 mentions)
Rabbit anti e cadherin, by Cell Signaling Technology (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
Rabbit anti cyclin d1, by Cell Signaling Technology (1 mentions)
Rabbit anti lrp6, by Santa Cruz Biotechnology (1 mentions)
5 protocols using odyssey infrared double fluorescence imaging system
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Metabolic Proteins
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For the Western blotting analysis, 80 μg of protein samples were separated on the sample buffer by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins were then transferred to nitrocellulose membranes. Membranes were blocked at room temperature for 1 h in 5% non-fat dry milk in PBS, followed by incubation with anti-HK2 (1:100 dilution), anti-Cyto C (1:50 dilution, Catalog No. ab13575; Abcam), anti-voltage dependent anion channel (VDAC) (1:1000 dilution, Catalog No. ab15895; Abcam), anti-cleaved Casp 3 (1:50 dilution, Catalog No. ab2302; Abcam), and anti-β-actin (1:6000 dilution, Catalog No. a0516; Sigma-Aldrich) or anti-GAPDH (1:6000 dilution, Catalog No. G9454; Sigma-Aldrich) antibodies overnight at 4°C. After incubated with secondary antibodies, the protein bands were visualized using the LI-COR Odyssey infrared double-fluorescence imaging system (LI-COR Biosciences, Lincoln, Nebraska, USA). The relative density value of each target protein band was normalized to the density of the corresponding β-actin, VDAC, or GAPDH band.
3
Western Blot Analysis of Protein Targets
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Total cell lysates were obtained using RIPA buffer (Solarbio, Beijing, China) containing 1:100 phenylmethylsulfonyl fluoride and phosphatase inhibitors. Cell lysates were resuspended in protein loading buffer containing 5 % mercaptoethanol. The proteins were separated by 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA) using a Mini-Protean electrophoresis module assembly (Bio-Rad) at 80 mV and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) for 100 min using the Mini Trans-Blot electrophoresis transfer cell (Bio-Rad) at 300 mA. The membranes were treated with IRDyeTM800 (green)- or IRDyeTM700 (red)-conjugated affinity purified anti-rabbit or anti-mouse IgG (LI-COR, Lincoln, NE, USA). Positive bands were visualized, and the intensity of the bands was evaluated using a LI-COR Odyssey infrareddouble-fluorescence imaging system (LI-COR). The primary antibodies used were anti-human USP13 (ab99421, Abcam), PTEN (ab32199, Abcam), and β-actin (CB100997M, California Bioscience, Coachella, CA, USA).
4
Western Blot Analysis of Wnt Signaling Pathway
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Total cell lysates were obtained using radioimmunoprecipitation assay buffer (Solarbio, Beijing, People’s Republic of China) containing 1:100 phenylmethylsulfonyl fluoride and phosphatase inhibitors. Total cellular proteins were extracted and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad Laboratories Inc., Hercules, CA, USA) and transferred to 0.22 μm nitrocellulose (NC) membranes (Sigma-Aldrich Co., St Louis, MO, USA). The membranes were blocked with 5% nonfat milk at room temperature for 2 hours and then incubated overnight at 4°C with rabbit anti-FZD8 (Sigma-Aldrich Co.), rabbit anti-LRP6 (Santa Cruz Biotechnology Inc., Dallas, TX, USA), rabbit anti-Axin1 (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-c-myc (Cell Signaling Technology), rabbit anti-cyclin D1 (Cell Signaling Technology), rabbit anti-β-catenin (Sigma-Aldrich Co.), rabbit anti-E-cadherin (Cell Signaling Technology), or mouse anti-β-actin antibodies (California Bioscience, Coachella, CA, USA). After incubating with IRDyeTM800 (green)- or IRDyeTM700 (red)-conjugated affinity-purified antirabbit or antimouse IgG (LI-COR, Lincoln, NE, USA), specific bands were visualized. The intensity of protein bands was evaluated using a LI-COR Odyssey infrared double-fluorescence imaging system (LI-COR).
5
Western Blot Analysis of Collagen I
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Protein from harvested cells was separated on a SDS-PAGE gel and then transferred onto polyvinylidene difluoride membranes (Millipore, USA). After blocking, the membranes were incubated overnight at 4°C with rabbit polyclonal anti-collagen I primary antibody (Abcam, Cambridge, CA, USA) and mouse polyclonal anti-β-actin primary antibody (California Bioscience, Coachella, CA, USA). The membranes were then treated with IRDyeࡊ800 (green) or IRDyeࡊ700 (red) conjugated affinity purified anti-rabbit or anti-mouse IgG (LI-COR, NE, USA). The positive western bands were visualized using a LI-COR Odyssey infrared double-fluorescence imaging system (LI-COR).
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