Immunoblotting Protocol for Analyzing Cell Signaling Pathways

The cells for immunoblotting were prepared as we described previously (22 (link)). Briefly, Cells were solubilized in lysis buffer (1.0% NP-40, 50 mM HEPES, pH7.4, 150 mM NaCl), containing protease and phosphatase inhibitor (Thermo fisher scientific). Cell lysates were separated by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE), transferred to a Polyvinylidene difluoride (PVDF) membrane (Merck), and detected with the following antibodies using ECL substrate (Bio-Rad): rabbit anti-CCAAT enhancer binding protein (C/EBP)α anti-phospho-C/EBPα (Ser21), anti-phospho- C/EBPα (Thr221/226), anti-Erk1/2, anti-phospho- Erk1/2, anti-JNK, anti-phospho-JNK, anti-NF-κB(p65), anti-phospho-NF-κB(p65), mouse anti-IκBα (Cell Signaling Technology), mouse anti-IκBβ (Santa Cruz Biotechnology), mouse anti-βactin (Sigma-aldrich), horseradish peroxidase (HRP)-conjugated mouse IgG (Cell Signaling Technology), or rabbit IgG antibodies (Cell Signaling Technology). Digital images were obtained using an ImageQuant LAS4000 mini instrument (GE Healthcare). Densitometry was performed on scanned blots using the ImageQuant TL software program (GE Healthcare).

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Matsuba S., Ura H., Saito F., Ogasawara C., Shimodaira S., Niida Y, & Onai N. (2023). An optimized cocktail of small molecule inhibitors promotes the maturation of dendritic cells in GM-CSF mouse bone marrow culture. Frontiers in Immunology, 14, 1264609.

Publication 2023

protease and phosphatase inhibitor ECL substrate anti-phospho-JNK anti-NF-κB(p65), anti-phospho-NF-κB(p65), mouse anti-IκBα mouse anti-βactin rabbit IgG antibodies ImageQuant LAS4000 mini instrument

Antibodies Buffer Cell Densitometry Erk1 Hepes Horseradish peroxidase Igg antibodies Iκbα Membrane Mouse Nacl Nf κb p65 Np 40 Phosphatase Polyvinylidene difluoride Protease Protein c Rabbit Sds polyacrylamide gel electrophoresis Sodium dodecyl sulfate

Corresponding Organization : Kanazawa Medical University

Other organizations : Kanazawa Medical University Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Protein expression and phosphorylation levels of CCAAT enhancer binding protein (C/EBP)α, Erk1/2, JNK, NF-κB(p65), IκBα, and IκBβ

control variables

Cell lysis buffer composition (1.0% NP-40, 50 mM HEPES, pH7.4, 150 mM NaCl)
Presence of protease and phosphatase inhibitors in cell lysis buffer
SDS-PAGE for protein separation
Transfer of proteins to PVDF membrane
Primary antibodies used for detection (rabbit anti-CCAAT enhancer binding protein (C/EBP)α, anti-phospho-C/EBPα (Ser21), anti-phospho-C/EBPα (Thr221/226), anti-Erk1/2, anti-phospho-Erk1/2, anti-JNK, anti-phospho-JNK, anti-NF-κB(p65), anti-phospho-NF-κB(p65), mouse anti-IκBα, mouse anti-IκBβ, mouse anti-βactin)
Secondary antibodies used (HRP-conjugated mouse IgG, HRP-conjugated rabbit IgG)
ECL substrate for detection
Imaging and densitometry analysis using ImageQuant LAS4000 mini and ImageQuant TL software

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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