Immunoblotting and Immunofluorescence Assays

Unless indicated otherwise, all reagents were purchased from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Monoclonal mouse antibodies against phospho-p38 MAP- kinase, NFκB and GAPDH were from Cell Signaling Technology (Danvers, MA). Antibodies to phospho- JNK (Thr183/185), and lamin A/C were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody to NF-κB phospho (S536) p65 used for tissue array staining was from Abcam (Cambridge, MA). Alexafluor488-conjugated secondary anti-mouse IgG (H+L) antibody was from Molecular Probes (Eugene, OR). Horseradish peroxidase (HRP) conjugated secondary antibodies to mouse IgG (H+L) and rabbit IgG (H+L) were from BioRad (Berkeley, CA). Antibodies against the 40kDa (gelatin-binding) region of fibronectin were described previously 13 (link). The functional upstream domain (FUD) of streptococcus pyogenes adhesin F1 protein was described previously 14 (link). The TLR4 (TAK-242) and NF-κB (BAY11-7082) inhibitors were from EMD Millipore (Temecula, CA). Recombinant His-tagged fibronectin Type III domains were described previously 15 (link).

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Cho C., Horzempa C., Longo C.M., Peters D.M., Jones D.M, & McKeown-Longo P.J. (2020). Fibronectin in the Tumor Microenvironment Activates a TLR4-dependent Inflammatory Response in Lung Cancer Cells. Journal of Cancer, 11(11), 3099-3105.

Publication 2020

NF-κB (BAY11-7082)

Adhesin Anti igg Antibodies Antibody Bay11 7082 Fetal bovine serum Fibronectin Fibronectin type iii domains Gapdh Gelatin Horseradish peroxidase Igg antibodies Inhibitors Lamin a c Molecular probes Monoclonal antibodies Mouse Nf κb P38 map kinase Protein domain Rabbit Streptococcus pyogenes Tak 242 Tissue

Corresponding Organization :

Other organizations : Albany Medical Center Hospital, University of Wisconsin–Madison

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Variable analysis

independent variables

Inhibitors: TLR4 (TAK-242) and NF-κB (BAY11-7082)

dependent variables

Phosphorylation of p38 MAP-kinase, NFκB, and JNK (Thr183/185)
NF-κB phosphorylation (S536) p65
Fibronectin expression

control variables

Reagents purchased from Sigma, Hyclone, Cell Signaling Technology, Santa Cruz Biotechnology, and Abcam
Monoclonal mouse antibodies against phospho-p38 MAP-kinase, NFκB, GAPDH, phospho-JNK (Thr183/185), and lamin A/C
Alexafluor488-conjugated secondary anti-mouse IgG (H+L) antibody
Horseradish peroxidase (HRP) conjugated secondary antibodies to mouse IgG (H+L) and rabbit IgG (H+L)
Antibodies against the 40kDa (gelatin-binding) region of fibronectin
Functional upstream domain (FUD) of streptococcus pyogenes adhesin F1 protein
Recombinant His-tagged fibronectin Type III domains

positive controls

Not specified

negative controls

Not specified

Annotations

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