Protocol detail

Mtb Infection-Induced Signaling Pathways

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Macrophages were infected with Mtb for the indicated times. Cells were lysed with lysis buffer consisting of 10 mM Tris–HCl, 1 mM EDTA, 140 mM NaCl, 0.1% DOC, 0.1% SDS, and Triton X-100 containing complete protease inhibitor cocktail (Calbiochem, San Diego, CA, USA). Western blotting was performed as previously described (27 (link)). Anti-phospho-GSK3β, anti-IκBα, anti-cathepsin D, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-phospho-PI3K, anti-PI3K, anti-phospho-p38, anti-p38, anti-phospho-ERK1/2, anti-phospho-MEK1/2, anti-phospho-JNK1/2, anti-phospho-IκBα, and anti-IRAK-M were purchased from Cell Signaling Technology.

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Lee H.J., Ko H.J., Song D.K, & Jung Y.J. (2018). Lysophosphatidylcholine Promotes Phagosome Maturation and Regulates Inflammatory Mediator Production Through the Protein Kinase A–Phosphatidylinositol 3 Kinase–p38 Mitogen-Activated Protein Kinase Signaling Pathway During Mycobacterium tuberculosis Infection in Mouse Macrophages. Frontiers in Immunology, 9, 920.

Publication 2018

Anti-phospho-GSK3β anti-IκBα anti-cathepsin D anti-β-actin Anti-phospho-PI3K anti-PI3K anti-phospho-p38 anti-p38 anti-phospho-ERK1/2 anti-phospho-MEK1/2 anti-phospho-JNK1/2 anti-phospho-IκBα anti-IRAK-M

Actin Anti d Buffer Cathepsin Cathepsin d Cells Edta Erk1 Gsk3β Irak m Macrophages Mek1 Nacl Phospho Pi3k Protease inhibitor Tris Triton x 100

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Variable analysis

independent variables

Mtb infection
Infection time

dependent variables

Phosphorylation of GSK3β
Expression of IκBα
Expression of cathepsin D
Phosphorylation of PI3K
Expression of PI3K
Phosphorylation of p38
Expression of p38
Phosphorylation of ERK1/2
Phosphorylation of MEK1/2
Phosphorylation of JNK1/2
Phosphorylation of IκBα
Expression of IRAK-M

control variables

Lysis buffer composition (10 mM Tris–HCl, 1 mM EDTA, 140 mM NaCl, 0.1% DOC, 0.1% SDS, and Triton X-100 containing complete protease inhibitor cocktail)
Western blotting protocol (as previously described in ref. 27)

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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