Journal: The Journal of Cell Biology

Article Title: Localization of Mad2 to Kinetochores Depends on Microtubule Attachment, Not Tension

doi:

Figure Lengend Snippet: Affinity-purified anti-XMad2 antibodies recognize one 24-kD band in PtK 1 cells. Immunoblot analysis with the affinity-purified anti-XMad2 polyclonal antibodies. Left, middle, right lanes , whole PtK 1 cell; left lane , Coomassie blue–stained Immobilon; middle lane , immunoblot with anti-XMad2 antibodies; right lane , immunoblot with anti-XMad2 antibodies that were pre-incubated with recombinant XMad2. Position of molecular weight markers are indicated on left.

Article Snippet: Full-length XMad2 tagged with six histidines at the NH 2 terminus ( ) was expressed in Escherichia coli with Qiaexpress vector pQE10 (Qiagen Inc., Chatsworth, CA).

Techniques: Affinity Purification, Western Blot, Staining, Incubation, Recombinant, Molecular Weight

Journal: Scientific Reports

Article Title: Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice

doi: 10.1038/s41598-020-58568-5

Figure Lengend Snippet: Characterization of lysosomal Ca 2+ release via TRPML1 channel activation in CASMCs using GCaMP3-ML1. ( A ) The strategy of GCaMP3-TRPML1 (GCaMP3-ML1) fusion. GCaMP3 is fused to the N-terminus of TRPML1. ( B ) Representative micrographs showing the changes of GCaMP3 fluorescence upon bath application of ML-SA1 and ionomycin to GCaMP3-ML1-transfected CASMCs. ( C,D ) Rapid increases in GCaMP3 fluorescence was induced by ML-SA1 (measured as the change of GCaMP3 fluorescence ∆F over basal fluorescence F0; ∆F/F0) in CASMCs transfected with GCaMP3-ML1. Subsequent application of GPN (200 µM) induced smaller responses than ML-SA1. On the other hand, ML-SA1 induced small responses in GCaMP3-ML1-expressing CASMCs that had received an application of GPN. The application of ionomycin (1 µM) induced maximal responses. ( E ) ML-SA1 had no effects on GCaMP3 fluorescence in GCaMP3-ML1-expressing CASMCs after pre-treatment with carmofur (Carm, 1 µM) for 30 minutes, however geniestein (Gen, 20 µM) pretreatment enhanced ML-SA1-induced Ca 2+ release through TRPML1 channel, (n = 4). Data are shown as means ± SEM. GPN: glycyl-L-phenylalanine 2-naphthylamide; Ionom: ionomycin. *p < 0.05 vs. Ctrl-Vehl group. # p < 0.05 vs. pre-treatment with ML-SA1 group by two-way ANOVA followed by Duncan’s test.

Article Snippet: CASMCs were transfected with GCaMP3-ML1 plasmid via nucleofector technology developed by Lonza (Basel, Switzerland).

Techniques: Activation Assay, Fluorescence, Transfection, Expressing

Journal: Scientific Reports

Article Title: Arterial Medial Calcification through Enhanced small Extracellular Vesicle Release in Smooth Muscle-Specific Asah1 Gene Knockout Mice

doi: 10.1038/s41598-020-58568-5

Figure Lengend Snippet: Effect of Ac gene deletion on lysosomal Ca 2+ release via TRPML1 channel activation in CASMCs. ( A ) ML-SA1 (20 µM) induced remarkable increases in GCaMP3 fluorescence in WT/WT CASMCs transfected with GCaMP3-ML1, which was decreased with Ac gene deletion in Asah1 fl/fl SM Cre CASMCs. ( B ) Summarized data shown in bar graph, (n = 5) ( C ) Sph (20 µM) induced remarkable increases in GCaMP3 fluorescence in WT/WT CASMCs transfected with GCaMP3-ML1. Also, Sph markedly increased GCaMP3 fluorescence in GCaMP3-ML1-expressing Asah1 fl/fl SM Cre CASMCs. The application of ionomycin (1 µM) induced maximal responses. ( D ) Summarized data showing responses in CASMCs transfected with GCaMP3-ML1 induced by Sph, (n = 4). Data are shown as means ± SEM. Ctrl: control; Sph: sphingosine; Ionom: ionomycin. *p < 0.05 vs. WT/WT Ctrl group. # p < 0.05 vs. WT/WT ML-SA1 group by two-way ANOVA followed by Duncan’s test and one-way ANOVA followed by Tukey’s test.

Article Snippet: CASMCs were transfected with GCaMP3-ML1 plasmid via nucleofector technology developed by Lonza (Basel, Switzerland).

Techniques: Activation Assay, Fluorescence, Transfection, Expressing