Journal: PLoS ONE

Article Title: Mechanisms Contributing to Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes versus Melanoma Cells

doi: 10.1371/journal.pone.0124154

Figure Lengend Snippet: Remaining expression levels (%) of PAX3 downstream target genes in melanocytes (NHEM-n and NHEM-a (I)) and melanoma cells (WM115 and M14) assessed two day post-transfection with PAX3 siRNA. Cells were treated with 10 μM siPAX3#1 (s224172, Ambion) alone, with consistent results observed across several independent experiments. Expression levels of downstream target genes were normalised to 18S (ΔCt) and calculated relative to the negative control siRNA (ΔΔCt). Each silencing experiment was performed with a biological duplicate.

Article Snippet: Additionally, immunoprecipitation was performed with anti-acetyl lysine, anti-phosphoserine and anti-ubiquitin antibodies individually, and anti-PAX3 antibody (mouse monoclonal, DSHB) was then used to probe the membranes ( ).

Techniques: Expressing, Transfection, Negative Control

Journal: PLoS ONE

Article Title: Mechanisms Contributing to Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes versus Melanoma Cells

doi: 10.1371/journal.pone.0124154

Figure Lengend Snippet: A) A comparison of PAX3E expression levels between melanocytes (NHEM-n and NHEM-a) and MM540 and M14 melanoma cell lines measured as number of reads per kilobase of exon model per million mapped fragments. Asterisks (*, **) denote significant differences. B) Immunoprecipitation with Pierce Direct IP Kit, using anti-PAX3 Ab (rabbit monoclonal, Life Technologies and immunoblotting with anti-acetyl-lysine, anti-phosphoserine and anti-ubiquitin. C) Immunoprecipitation (with IgG beads) using anti-phosphoserine, anti-acetylysine, anti-ubiquitin and anti-PAX3 (rabbit polyclonal, Abcam), immunoblotted with anti-PAX3 (mouse monoclonal, DSHB) in human epidermal melanocytes (NHEM-n and NHEM-a (I)) and WM115 and M14 melanoma cell lines.

Article Snippet: Additionally, immunoprecipitation was performed with anti-acetyl lysine, anti-phosphoserine and anti-ubiquitin antibodies individually, and anti-PAX3 antibody (mouse monoclonal, DSHB) was then used to probe the membranes ( ).

Techniques: Comparison, Expressing, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Mechanisms Contributing to Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes versus Melanoma Cells

doi: 10.1371/journal.pone.0124154

Figure Lengend Snippet: A) PAX3 protein levels relative to β-actin. Each sample was run in duplicate on separate gels and the density of each band was assessed by densitometric scanning using the GS-800 Calibrated Densitometer (Bio-Rad). The density of each PAX3 band was compared to that of β-actin for each lane on the same membrane and the fold change calculated and graphed. The error bars represent the standard deviation between duplicate samples. B) PAX3 mRNA expression in neonatal and adult normal human epidermal melanocytes and primary and metastatic melanoma cell lines. RT-qPCR was performed using RNA from neonatal (NHEM-n) and adult (NHEM-a (P) and NHEM-a (I)) human epidermal melanocytes as well as six primary (MM200, MM229, MM329, MM540, MM622 and WM115) and five metastatic (A2058, M14, SKMEL2, SKMEL5 and UACC62) melanoma cell lines. The level of PAX3 expression was calculated as fold-change relative to GAPDH expression levels. All PCRs were performed in triplicate and average values were used to calculate fold change over GAPDH for each sample. All samples were run in biological duplicates and the error bars represent the standard deviation of the biological replicates. C) Immunocytochemistry of neonatal and adult human epidermal melanocytes, five metastatic melanoma cell lines and six primary melanoma cell lines. Nuclear PAX3 protein expression (red) is observed in all melanoma cell lines and primary melanocytes. Cell nuclei are stained with DAPI (blue). The scale measures 100 μM. A negative control was included with each experimental run and proved negative in each instance.

Article Snippet: Additionally, immunoprecipitation was performed with anti-acetyl lysine, anti-phosphoserine and anti-ubiquitin antibodies individually, and anti-PAX3 antibody (mouse monoclonal, DSHB) was then used to probe the membranes ( ).

Techniques: Membrane, Standard Deviation, Expressing, Quantitative RT-PCR, Immunocytochemistry, Staining, Negative Control

Journal: PLoS ONE

Article Title: Mechanisms Contributing to Differential Regulation of PAX3 Downstream Target Genes in Normal Human Epidermal Melanocytes versus Melanoma Cells

doi: 10.1371/journal.pone.0124154

Figure Lengend Snippet: A) PAX3 , MITF and BRN2 mRNA expression levels in melanocytes and melanoma cells. RT-qPCR was performed to assess expression levels of PAX3 , MITF and BRN2 mRNA in primary melanocytes and melanoma cell lines. All PCRs were performed in triplicate and expression values were calculated as fold change relative to GAPDH . Biological replicates were performed for each sample and error bars denote the standard deviation between biological replicates. B) PAX3, MITF and BRN2 protein levels in primary melanocytes and melanoma cells. Total protein from primary melanocytes and melanoma cells was separated by SDS-PAGE and immunoblotted with anti-PAX3, anti-MITF, anti-BRN2 and anti-GAPDH.

Article Snippet: Additionally, immunoprecipitation was performed with anti-acetyl lysine, anti-phosphoserine and anti-ubiquitin antibodies individually, and anti-PAX3 antibody (mouse monoclonal, DSHB) was then used to probe the membranes ( ).

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, SDS Page

Journal: Bio-Medical Materials and Engineering

Article Title: Expression of Cx43 and Pax3 proteins in the human placental villi and decidua during early pregnancy

doi: 10.3233/bme-141214

Figure Lengend Snippet: Fig. 5. Pax3 protein expression in the human placental trophoblast at 5 weeks of pregnancy, SABC×400. Fig. 6. Pax3 protein expression in the human placental trophoblast at 7 weeks of pregnancy, SABC×400.

Article Snippet: Anti-Cx43 and anti-Pax3 polyclonal antibodies and the accessory kits were all purchased from Wuhan Boster Biological Technology Co., Ltd.

Techniques: Expressing

Journal: Bio-Medical Materials and Engineering

Article Title: Expression of Cx43 and Pax3 proteins in the human placental villi and decidua during early pregnancy

doi: 10.3233/bme-141214

Figure Lengend Snippet: Fig. 7. Pax3 protein expression in the human decidual cells at 5 weeks of pregnancy, SABC×400. Fig. 8. Pax3 protein expression in the human decidual cells at 7 weeks of pregnancy, SABC×400.

Article Snippet: Anti-Cx43 and anti-Pax3 polyclonal antibodies and the accessory kits were all purchased from Wuhan Boster Biological Technology Co., Ltd.

Techniques: Expressing