Reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Antibodies for mTOR, Rictor, UCHL1, phospho-NF-kB p65, phospho-AktSer473 were purchased from Cell Signaling Technologies (Danvers, MA), β-actin and NF-kB from Invitrogen (Carlsbad, CA), goat anti-mouse IgG (Horseradish Peroxidase) from Jackson ImmunoResearch Laboratories. The anti-human goat NAMPT pAb was custom-generated as previously described10 (link),11 (link). The eNAMPT-neutralizing humanized mAb was provided by Aqualung Therapeutics (Tucson, AZ) as previously described11 (link). See Supplemental Materials and Methods for more details.
Goat anti mouse igg horseradish peroxidase
Manufactured by Jackson ImmunoResearch
Goat anti-mouse IgG (Horseradish Peroxidase) is a secondary antibody conjugated with the enzyme Horseradish Peroxidase. It is used to detect and quantify mouse immunoglobulin G (IgG) in various immunological assays.
Automatically generated - may contain errors
Lab products found in correlation
Phospho nf kb p65, by Cell Signaling Technology (1 mentions)
Uchl1, by Cell Signaling Technology (1 mentions)
Rictor, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
T per lysis buffer, by Thermo Fisher Scientific (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Anti actin, by Merck Group (1 mentions)
Sc 8418, by Santa Cruz Biotechnology (1 mentions)
A4700, by Merck Group (1 mentions)
Anti phospho s6, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
2 protocols using goat anti mouse igg horseradish peroxidase
1
Quantifying Cellular NAMPT Signaling
2
Kidney Protein Analysis by Western Blot
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Kidneys were collected and homogenized in T-PER lysis buffer (Thermo Fisher Scientific) supplemented with protease (Pierce) and phosphatase inhibitors (Roche). The homogenized lysate was incubated at 4°C for 20 min and cell debris cleared by centrifugation at 12,000 g for 10 min. 50 µg of protein lysate from each kidney was analyzed by SDS-PAGE. Membranes were incubated overnight at 4°C with the following primary antibodies: anti–phospho–p-S6K (1:200; Tyr 389; 9205; Cell Signaling Technology), anti–p-S6K (1:1,000; sc-8418; Santa Cruz Biotechnology), anti–phospho-S6 (1:200; Ser235/236; 2211; Cell Signaling Technology), anti-S6 (1:500; sc-74459; Santa Cruz Biotechnology), and anti-actin (1:1,000; A4700; Sigma-Aldrich). Secondary antibodies included donkey anti–rabbit IgG–horseradish peroxidase (Sigma-Aldrich) and goat anti–mouse IgG–horseradish peroxidase (Jackson ImmunoResearch Laboratories). Blots were imaged on a C-DiGit Blot Scanner (LI-COR), and signal intensities for each protein were quantified and normalized to actin.
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