ppak1 plasmid  (Addgene inc)

Journal: Cancers

Article Title: The PAK1-Stat3 Signaling Pathway Activates IL-6 Gene Transcription and Human Breast Cancer Stem Cell Formation

doi: 10.3390/cancers11101527

Figure Lengend Snippet: PAK1 physically interacts with Stat3, PAK1 and Stat3 colocalize in the nucleus and PAK1 inactivation induces Stat3 dephosphorylation. ( A ) Total protein from breast cancer cells was immunoprecipitated with anti-PAK1 or anti-Stat3 antibodies, followed by immunoblotting using anti-Stat3 or anti-PAK1 antibodies. ( B ) Immunofluorescence analysis of PAK1 (green) and Stat3 (red) expression in breast cancer cells using the indicated antibodies. Images were captured by microscopy at 10× magnification. ( C ) Effect of ivermectin on the pStat3 level. ( D ) Immunofluorescence analysis of PAK1 (green) and Stat3 (red) expression levels in breast cancer cells after ivermectin treatment. Images were captured by microscopy at 10× magnification (scale bar = 100 μm).

Article Snippet: The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Expressing, Microscopy

Journal: Cancers

Article Title: The PAK1-Stat3 Signaling Pathway Activates IL-6 Gene Transcription and Human Breast Cancer Stem Cell Formation

doi: 10.3390/cancers11101527

Figure Lengend Snippet: Effect of a JAK2 inhibitor on nuclear PAK1 and Stat3 levels and on the colocalization of PAK1 and Stat3. ( A ) Immunofluorescence analysis of PAK1 (green) and Stat3 (red) expression in breast cancer cells using anti-PAK1 and anti-Stat3 antibodies after TG101209 treatment. Images were captured by microscopy at 10× magnification. ( B ) Effect of TG101209 on cytosolic and nuclear pStat3 protein levels.

Article Snippet: The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Techniques: Immunofluorescence, Expressing, Microscopy

Journal: Cancers

Article Title: The PAK1-Stat3 Signaling Pathway Activates IL-6 Gene Transcription and Human Breast Cancer Stem Cell Formation

doi: 10.3390/cancers11101527

Figure Lengend Snippet: PAK1 affects IL-6 gene expression through Stat3-mediated regulation. ( A ) Electrophoretic mobility shift assay (EMSA) using mammosphere nuclear proteins. Nuclear proteins were incubated with an IRDye 800-Stat probe at room temperature and separated by 6% native PAGE. Lane 1: stat probe; lane 2: nuclear proteins with stat probe; lane 3: 10-fold concentration of the self-competitor oligo; lane 4: nuclear proteins incubated with a 10-fold concentration of the mutated-Stat3 probe. The arrow indicates the DNA/Stat3 complex in mammosphere nuclear lysates. ( B ) EMSA analysis of PAK1 and Stat3 binding to the Stat3 DNA-binding sequence. Lane 1: Stat probe only; lane 2: nuclear proteins with Stat probe; lane 3: 10-fold concentration of the self-competitor oligo; lane 4: 10-fold concentration of the mutated Stat probe; lane 5: pStat3 antibody; lane 6: PAK1 antibody; lane 7: pStat3 and PAK1 antibody; lane 8: IgG. The arrow indicates the Stat3/PAK1/DNA complex in nuclear proteins. ( C ) ChIP (chromatin immunoprecipitation) analysis of Stat3 and PAK1 recruitment to the IL-6 gene promoter region in breast cancer cells. Double-ChIP analysis of the Stat3-PAK1/DNA complex binding to the human IL-6 gene promoter in breast cancer cells. The first ChIP was performed with an anti-pStat3 or anti-PAK1 antibody, followed by a second ChIP with an anti-PAK1 or anti-pStat3 antibody, respectively. Input represents 1% of the sonicated DNA. ( D ) RT-qPCR analysis of human IL-6 mRNA levels in breast cancer cells transiently overexpressing PAK1 and Stat3 genes. The relative mRNA expression levels are presented. The data are presented as the mean ± SD; n = 3; * p < 0.05 vs. control vector. ( E ) Nuclear levels of Stat3 and NF-κB (p65) protein in mammospheres were examined with antibodies against Stat3 and p65. Ivermectin treatment reduced nuclear pStat3 levels in the mammospheres. ( F ) EMSA of lysates of mammospheres treated with ivermectin. Nuclear proteins were treated with an IRDye 800-Stat sequence probe and separated by 6% native PAGE. Lane 1: stat probe; lane 2: nuclear proteins with stat probe; lane 3: ivermectin-treated proteins with stat probe; lane 4: 10-fold concentration of self-competitor oligo; lane 5: 10-fold concentration of mutated Stat3 oligo. Ivermectin treatment decreases the DNA/Stat3 interaction in mammosphere nuclei. ( G ) Transcriptional levels of the human IL-6 gene were assayed in ivermectin-treated cells using IL-6 -specific primers. β-actin expression was used to normalize the values. The data are presented as the mean ± SD; n = 3; * p < 0.05 vs. control. ( H ) Immunoblot analysis of culture medium from mammospheres using an IL-6 antibody and equal numbers of mammospheres as a control. Ivermectin treatment reduced extracellular IL-6 levels in mammosphere culture medium. ( I ) The cytokine profiles of conditioned media from mammosphere cultures were determined with cytokine-specific antibodies and cytokine beads. Ivermectin reduced extracellular IL-6 levels in the mammosphere cultures.

Article Snippet: The pPAK1 and pStat3 plasmids were purchased from Addgene (Watertown, MA, USA).

Techniques: Gene Expression, Electrophoretic Mobility Shift Assay, Incubation, Clear Native PAGE, Concentration Assay, Binding Assay, Sequencing, Chromatin Immunoprecipitation, Sonication, Quantitative RT-PCR, Expressing, Control, Plasmid Preparation, Western Blot