protein expression  (Addgene inc)

Journal: International Journal of Molecular Sciences

Article Title: GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells

doi: 10.3390/ijms25063181

Figure Lengend Snippet: Citrullination does not alter the number of MT comets. ( A ) LβT2 cells were transfected with the EB1-GFP plasmid for 48 hr, pre-treated with DMSO or 1 μm BB-ClA for 12 h, serum starved, and then stimulated with 10 nM GnRHa for 0 and 30 min. The cells were then placed in a Tokai Hit-STX Stage Top Incubator at 37 °C with 5% CO 2 , and images were captured at 0.5 s intervals for 65–94 s by an SDCM using a 60X oil objective. Image stacks were used to analyze the number of EB1 comets passing through 10 μm long sections ( n = 3 sections) in each cell ( n = 10 cells; n = 30 sections total); see the representative line overlays in the images. Line colors indicate different treatment groups (Red = Vehicle, Blue = GnRHa 30, Purple = BB-ClA, Yellow = GnRHa 30 + BB-ClA). Kymographs (bottom row) show EB1-GFP comets passing transversely through the lines depicted in the corresponding images of cells (top row), with time on the vertical axis and distance on the horizontal axis. ( B ) The bar graph depicts quantitative analysis of the fold change in the flux of EB1-GFP comets, in comets per second, through the 10 μm sections. Each circle represents an individual comet (Red = Vehicle, Blue = GnRHa 30, Green = BB-ClA, Orange = GnRHa 30 + BB-ClA). The difference between the means of each group were separated using Tukey’s HSD and error bars represent the SEM.

Article Snippet: The Human EB1 GFP vector (JB131) was a gift from Tim Mitchison and Jennifer Tirnauer (Addgene plasmid # 39299; http://n2t.net/addgene:39299 (accessed on 29 March 2021); RRID: Addgene_39299).

Techniques: Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells

doi: 10.3390/ijms25063181

Figure Lengend Snippet: Citrullination does not alter polymerization speed but contributes to GnRHa-induced MT lifetime. ( A ) LβT2 cells were transfected with the EB1-GFP plasmid for 48 hr, pre-treated with DMSO or 1 μm BB-ClA for 12 h, serum starved, and then stimulated with 10 nM GnRHa for 0 and 30 min. The cells were then placed in a Tokai Hit-STX Stage Top Incubator at 37 °C with 5% CO 2 , and live cell images were captured at 0.5 s intervals for 65–94 s by an SDCM using a 60X oil objective. Images are kymographs generated in Fiji software version 2.14.0/1.54f showing MT tracks running parallel (or along) the kymograph’s distance axis. Line colors indicate different treatment groups (Red = Vehicle, Blue = GnRHa 30, Purple = BB-ClA, Yellow = GnRHa 30 + BB-ClA). Bar graphs depict the quantitative analysis of track polymerization speeds ( B ) and track lifetimes ( C ) ( n = 30) measured in Fiji software version 2.14.0/1.54f using Rietdorff’s kymograph plugin. Each circle represents an individual observation (Red = Vehicle, Blue = GnRHa 30, Green = BB-ClA, Orange = GnRHa 30 + BB-ClA). The difference between the means of each group were separated using Tukey’s HSD. Asterisks indicate a significant difference between groups (*** = p < 0.001) and error bars represent the SEM.

Article Snippet: The Human EB1 GFP vector (JB131) was a gift from Tim Mitchison and Jennifer Tirnauer (Addgene plasmid # 39299; http://n2t.net/addgene:39299 (accessed on 29 March 2021); RRID: Addgene_39299).

Techniques: Transfection, Plasmid Preparation, Generated, Software