Lung tumours (≥2 mm in diameter) were freshly dissected from male and female Kras/Lkb1 mice at 8–10 weeks post Ad-Cre treatment. These tumours were immediately cut into several pieces, one piece was used for HE and IHC inspection, and the rest pieces were snap-frozen separately for western blotting and real-time PCR analyses. Western blot assays were performed as previously described with antibodies against p63 (SC-8431, Santa Cruz, this antibody specifically recognizes DNp63; 1:200, 1 ng μl−1), Lox (L4669, Sigma; 1:800, 1.25 ng μl−1), SP-C (AB3786, Chemicon; 1:2,000) and Actin (C-1, Santa Cruz; 1:2,000, 0.1 ng μl−1). Total RNA and genomic DNA were prepared as previously described35 (link). Total RNA was retrotranscribed into first-strand cDNA using RevertAid First Strand cDNA Synthesis Kit (Fermentas). The cDNAs were then used for real-time PCR on a 7500 Fast Real-Time PCR System (Applied Biosystems) using SYBR-Green Master PCR mix (Toyobo). β-actin served as internal control. The primers for PCR were summarized in Supplementary Information (Supplementary Table 5 ).
L4669
Manufactured by Merck Group
Sourced in United States
The L4669 is a laboratory equipment product from Merck Group. It is a precision instrument designed for scientific applications. The core function of the L4669 is to provide accurate measurements and data collection for research and analysis purposes. No further details about the specific intended use or features of the product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Sc 8431, by Santa Cruz Biotechnology (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Toyobo (1 mentions)
Ab3786, by Merck Group (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
Nb110 41568, by Novus Biologicals (1 mentions)
Amicon ultra cel 10, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Ab96233, by Abcam (1 mentions)
Jla20, by Merck Group (1 mentions)
2 protocols using l4669
1
Kras/Lkb1 Mouse Lung Tumor Analysis
2
Protein Concentration and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Secreted proteins were first concentrated using Amicon Ultra Cel-10 centrifugal filters (MWCO 10.000; Millipore, Bedford, MA, USA) as previously described [94 (link)]. Cellular lysates or secreted proteins were resolved by 10% SDS-PAGE, and the proteins were electrophoretically transferred onto nitrocellulose membrane (Bio-Rad, Herwles, Hercules, CA, USA) and analyzed as previously [24 (link)]. Secreted and cellular protein bands of human melanoma cells and fibroblasts were quantified using Image Studio Lite (LI-COR Biotechnology, Lincoln, NE, USA). A rabbit polyclonal antibody recognizing the C-terminal domain of LOX (L4669; Sigma-Aldrich), a rabbit polyclonal LOX propeptide antibody to LOX-PP (NB110-41568; Novus Biologicals, Littleton, CO, USA), and a rabbit polyclonal antibody to LOXL2 (ab96233; Abcam, Cambridge, UK) were used to detect the respective proteins. Secreted protein samples were verified to contain comparable amounts of total proteins by silver staining. Mouse monoclonal antibodies to alpha-tubulin (DM1A; Abcam) and actin (JLA20; Merck, Millipore, Billerica, MA, USA) were used as loading controls for cellular proteins.
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