The cDNA sequences for genes were synthesized by Wuhan Gene Create Biological Engineering Co., Ltd. (Wuhan, China). The cDNA for each gene was PCR-amplified and inserted into pAdTrack-CMV previously digested with BglII/HindIII using the In-Fusion HD cloning kit (Takara, Japan) (Sleight et al., 2010 (link)). The resulting plasmid was linearized with PmeI, and then homologous recombination was performed with pAdeasy-1 in E. coli BJ5183. The recombinant plasmid with PacI linearization was used to transfect AAV-293 cells to produce recombinant adenovirus particles using the Advanced DNA RNA transfection reagent (Zeta Life, Menlo Park, CA, United States). RT-qPCR (Nolan et al., 2006 (link)) and Western-blot (following the Elabscience Western blot detection kit operation manual) were used to detect the expression of target gene. The canine ADMSCs were transfected with the recombinant adenovirus particles.
Western blot detection kit
Manufactured by Elabscience
The Western blot detection kit is a set of reagents and tools used to detect and quantify specific proteins in a sample. It provides the necessary components to perform the Western blot technique, which is a widely used analytical method in molecular biology and biochemistry.
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Lab products found in correlation
Advanced dna rna transfection reagent, by Zeta Life (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Anti pkcα, by Abcam (1 mentions)
Anti pkcγ, by Abcam (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti pkcδ, by Abcam (1 mentions)
Anti pkc, by Abcam (1 mentions)
Anti glp 1r, by Abcam (1 mentions)
Anti p akt, by Abcam (1 mentions)
Anti pi3k, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit lgg, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Cytiva (1 mentions)
Anti akt, by Abcam (1 mentions)
2 protocols using western blot detection kit
1
Adenovirus-Mediated Gene Delivery to Canine ADMSCs
2
Westernblot Analysis of Cardiac Proteins
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Both H9C2 cells and myocardial tissues were harvested for Western blot analysis (Western Blot Detection Kit, Elabscience) following standard protocol according to the manufacturer’s instructions and Towbin system buffer was used. Primary antibodies used in Western blot were as follows: anti-GLP-1R (1:200, Abcam, UK), anti-Akt (1:200, Abcam, UK), anti-p-Akt (1:200, Abcam, UK), anti-PI3K (1:500, Santa Cruz, USA), anti-PKC (1:500, Abcam, UK), anti-PKCα (1:500, Abcam, UK), anti-PKCβ (1:500, Abcam, UK), anti-PKCγ (1:500, Abcam, UK), anti-PKCδ (1:500, Abcam, UK) and anti-β-actin (1:500, Santa Cruz, USA). Secondary antibodies were horseradish peroxidase-conjugated goat anti-rabbit lgG (Santa Cruz, USA) at 1:5000 dilution. The results were visualized using an enhanced chemiluminescence system (ECL, Amersham). Densitometric analysis was conducted using ImageJ software (NIH, USA).
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