Bone marrow from naïve mice was collected as described above. 1×106 cells were cultured in a 12-well flat-bottom plate and incubated for three days in RPMI containing 10% serum. Supernatant with non-adherent cells was removed. After 4h incubation (which was optimal for mTOR phosphorylation) with or without 5 μg/mL β-glucan ± 100 ng/mL IL-38 in RPMI, cells were lysed in RIPA buffer containing Protease Inhibitor (Roche). Total protein content was determined by BCA assay (Thermo Fisher Scientific) and equal amounts of proteins were subjected to SDS-PAGE on pre-casted 4–15% gels (Biorad, CA, USA). The separated proteins were transferred to a nitrocellulose membrane (Biorad), which was blocked in 5% BSA (Sigma) and incubated overnight at 4oC with rabbit polyclonal antibodies against (phospho) mTOR, Akt, 4EBP1, and S6K (Cell Signaling, Danvers, MA, USA), and β-actin (Sigma), which were visualized using a polyclonal secondary antibody (Dako, Belgium) and SuperSignal West Femto Substrate (Thermo Fisher Scientific) for the phosphorylated proteins or ECL substrate for the other proteins (Biorad).
Polyclonal secondary antibody
Manufactured by Agilent Technologies
Sourced in Belgium
Polyclonal secondary antibodies are laboratory reagents used to detect and quantify target proteins in various experimental techniques, such as Western blotting, immunohistochemistry, and ELISA. They are produced by immunizing animals with a specific antigen, resulting in the generation of a diverse population of antibodies that recognize multiple epitopes on the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west femto substrate, by Thermo Fisher Scientific (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
β actin, by Merck Group (2 mentions)
Pre casted 4 15 gels, by Bio-Rad (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4e bp1, by Cell Signaling Technology (1 mentions)
2 protocols using polyclonal secondary antibody
1
Bone Marrow Cell Signaling Assay
2
Activation of mTOR Signaling by β-Glucan
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Bone marrow from naïve mice was collected as described earlier. A total of 1 × 106 cells were cultured in a 12‐well flat‐bottom plate and incubated for 3 d in RPMI containing 10% serum. Supernatant with nonadherent cells was removed. After 4 h incubation (which was optimal for mTOR phosphorylation) with or without 5 μg/ml β‐glucan ± 100 ng/ml IL‐38 in RPMI, cells were lysed in RIPA buffer containing protease inhibitor (Roche, Basel, Switzerland). Total protein content was determined by BCA assay (Thermo Fisher Scientific), and equal amounts of proteins were subjected to SDS‐PAGE on precasted 4–15% gels (Biorad, Hercules, CA, USA). The separated proteins were transferred to a nitrocellulose membrane (Biorad), which was blocked in 5% BSA (Sigma) and incubated overnight at 4°C with rabbit polyclonal antibodies against (phospho) mTOR, Akt, 4EBP1, and S6K (Cell Signaling, Danvers, MA, USA), and β‐actin (Sigma), which were visualized using a polyclonal secondary antibody (Dako, Leuven, Belgium) and SuperSignal West Femto Substrate (Thermo Fisher Scientific) for the phosphorylated proteins or ECL substrate for the other proteins (Biorad).
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