monoclonal mouse anti alpha tubulin primary antibody  (Millipore)

Journal: Journal of young investigators

Article Title: Larger sperm size may contribute to reproductive isolation between Etheostoma species

doi:

Figure Lengend Snippet: The nucleus can be identified by DAPI staining (upper left) and the tail can be identified by the alpha-tubulin Alexa 488 staining (upper right). The bottom right is the composite; the bottom left shows a bright field image.

Article Snippet: Anti-alpha-tubulin (bovine) mouse IgG1 monoclonal primary antibody (Life Technologies, Eugene, OR) was used to stain tubulin proteins abundant in sperm flagellum.

Techniques: Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: Alanine Scanning Mutagenesis of the C-Terminal Cytosolic End of Gpm6a Identifies Key Residues Essential for the Formation of Filopodia

doi: 10.3389/fnmol.2018.00314

Figure Lengend Snippet: Alanine scanning mutagenesis of the C-terminal cytosolic end of Gpm6a. (A) Amino acid sequence of the C-terminal end (aa 241–278) of the mouse Gpm6a (NCBI Accession: NP_705809.1). Charged amino acids substituted with alanine are highlighted in red. Blue lines indicate charged residues mutated simultaneously. Residues that affect filopodium formation when mutated to alanine are indicated by yellow boxes. (B) N2a cells transfected with the indicated vectors and labeled with rhodamine red-conjugated phalloidin to visualize F-actin cytoskeleton were analyzed by fluorescence microscopy. The percentage of transfected N2a cells showing filopodia was quantified in red channel visualizing rhodamine red-phalloidin. On average, 97–125 cells for each transfection condition done in duplicates were analyzed. Data are means ± SEM. One-way ANOVA followed by Dunnett’s multiple comparison test for post hoc effects, ∗ p < 0.05 EGFP vs R247A-EGFP, ∗ p < 0.05 EGFP vs K257A-EGFP, ∗ p < 0.05 EGFP vs E261A/D264A-EGFP, ∗ p < 0.05 EGFP vs H266A/R269A-EGFP, ∗ p < 0.05 EGFP vs K271A/E272A/R273A-EGFP, ∗∗ p < 0.01 EGFP vs Gpm6a wt-EGFP, ∗∗ p < 0.01 EGFP vs E252A-EGFP, ∗∗ p < 0.01 EGFP vs H263A-EGFP, ∗∗∗ p < 0.001 EGFP vs K243A-EGFP, ∗∗∗ p < 0.001 EGFP vs D244A-EGFP. One-way ANOVA revealed no significant differences for EGFP vs K250-EGFP, EGFP vs D253A/K255A-EGFP, and EGFP vs E258A/E259A-EGFP. (C) Localization of K250-EGFP, D253A/K255A-EGFP, and E258A/E259A-EGFP mutants in N2a cells. Confocal images of N2a cells transfected with the indicated vectors and labeled with rhodamine red-conjugated phalloidin. Gpm6a wt-EGFP and EGFP alone were used as controls. K250A, D253A/K255A, and E258A/E259A localize to the plasma membrane of N2a but fail to form filopodial protrusions. Scale bar, 10 μm. (D) Western blot of lysates from N2a cells overexpressing the indicated constructs. Immunnoblot (IB) was analyzed using the rabbit anti-GFP antibody detected by the goat anti-rabbit secondary IRDye800 CW. Bands representing Gpm6a proteins are indicated by asteriscs. As a loading control alpha-tubulin was detected using the mouse anti-alpha-tubulin monoclonal antibody followed by the goat anti-mouse secondary IRDye680 LT.

Article Snippet: After 1 h of blocking in TBS containing 0.2% (vol/vol) Tween-20 and 2% (vol/vol) fish skin gelatin (FSG), the membranes were incubated with the rabbit anti-GFP polyclonal primary antibody (1/1000, Thermo Fisher Scientific) and the mouse anti-alpha-tubulin monoclonal primary antibody (1/2000, Sigma) overnight.

Techniques: Mutagenesis, Sequencing, Transfection, Labeling, Fluorescence, Microscopy, Western Blot, Construct

Journal: Frontiers in Molecular Neuroscience

Article Title: Alanine Scanning Mutagenesis of the C-Terminal Cytosolic End of Gpm6a Identifies Key Residues Essential for the Formation of Filopodia

doi: 10.3389/fnmol.2018.00314

Figure Lengend Snippet: Deletion of the C-terminal but not the N-terminal intracellular domain of Gpm6a interferes with filopodium formation in neuroblastoma cell line N2a. (A) Schematic structure of the EGFP-tagged wild type (wt) Gpm6a and Gpm6a deletion constructs: Gpm6a wt-EGFP (full length wt Gpm6a, 1–278 aa), Gpm6a ΔN-EGFP (Δ1–16), Gpm6a ΔC-EGFP (Δ243–278). The domain organization is indicated by colored boxes: the N-terminal and the C-terminal intracellular domains (red); the four transmembrane domains (TM1-4; gray); the intracellular (IC), the small extracellular (EC1), and the large extracellular (EC2) loops (white). (B) Western blot of lysates from N2a cells overexpressing the indicated constructs. Immunnoblot (IB) was analyzed using the rabbit anti-GFP antibody detected by the goat anti-rabbit secondary IRDye800 CW. Bands representing Gpm6a proteins are indicated by asteriscs. As a loading control alpha-tubulin was detected using the mouse anti-alpha-tubulin monoclonal antibody followed by the goat anti-mouse secondary IRDye680 LT. Below, the control Western blot of lysates from non-transfected N2a cells or N2a overexpressing EGFP alone shows the specificty of detected signal. (C) Localization of ΔN and ΔC Gpm6a mutants in N2a cells. Confocal images of N2a cells transfected with the indicated vectors and labeled with rhodamine red-conjugated phalloidin to visualize F-actin cytoskeleton. Gpm6a wt-EGFP and EGFP alone were used as controls. Gpm6a wt-EGFP and Gpm6a ΔN-EGFP accumulate at plasma membrane and in filopodial protrusions (second and third row). Gpm6a ΔN-EGFP and Gpm6a ΔC-EGFP show higher cytoplasmic localization comparing to the wt Gpm6a. Overexpression of Gpm6a ΔC-EGFP does not induce filopodia formation (bottom row). Scale bar, 10 μm. (D,E) The percentage of transfected N2a cells showing filopodia was quantified in red channel visualizing rhodamine red-phalloidin. On average, 97–119 cells for each transfection condition done in duplicates were analyzed in multiple experiments. Data are means ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test for post hoc effects. (D) Gpm6a ΔN-EGFP: ∗∗ p < 0.01 EGFP vs Gpm6a wt-EGFP, ∗∗ p < 0.01 EGFP vs Gpm6a ΔN-EGFP. (E) Gpm6a ΔC-EGFP: ∗∗ p < 0.01 EGFP vs Gpm6a wt-EGFP, ∗∗ p < 0.01 Gpm6a wt-EGFP vs Gpm6a ΔC-EGFP. No statistically significant differences between EGFP and Gpm6a ΔC-EGFP were detected.

Article Snippet: After 1 h of blocking in TBS containing 0.2% (vol/vol) Tween-20 and 2% (vol/vol) fish skin gelatin (FSG), the membranes were incubated with the rabbit anti-GFP polyclonal primary antibody (1/1000, Thermo Fisher Scientific) and the mouse anti-alpha-tubulin monoclonal primary antibody (1/2000, Sigma) overnight.

Techniques: Construct, Western Blot, Transfection, Labeling, Over Expression