Journal: Molecular Biology of the Cell

Article Title: SUMO-targeted ubiquitin ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin

doi: 10.1091/mbc.E15-12-0827

Figure Lengend Snippet: Slx5 prevents mislocalization of Cse4 to euchromatin. (A) Cse4 expressed from its own promoter is increased in chromatin fraction in psh1 ∆, slx5 ∆, and psh1 ∆ slx5 ∆ strains. Whole-cell extracts (WCEs) prepared from equal numbers of logarithmically growing cells in YPD were fractionated into soluble and chromatin fraction and assayed by Western blot analysis. Tub2 and histone H3 were used as markers for soluble and chromatin fractions, respectively. Two blots shown for Cse4 are indicated (L, long exposure; S, short exposure). (B, C) Cse4 expressed from its own promoter is mislocalized in psh1 ∆, slx5 ∆, and psh1 ∆ slx5 ∆ strains. Chromosome spreads were done by logarithmically growing cells in YPD. DAPI (blue) and α-HA (red) staining were used to visualize DNA and Cse4 localization, respectively. In wild-type strains, Cse4 is predominantly localized to one or two kinetochore clusters. In mutant strains, mislocalization of Cse4 is observed as multiple foci or diffused localization throughout the nucleus. The graph quantifies the number of cells exhibiting Cse4 mislocalization, and error bars are average deviation of two independent experiments. The number of cells used is indicated ( n ). Isogenic yeast strains used are wild type (YMB7290), psh1 ∆ (YMB7393), slx5 ∆ (YMB7588), and psh1 ∆ slx5 ∆ (YMB7607).

Article Snippet: Cells were visualized by DAPI staining (1 μg/ml in phosphate-buffered saline) mounted in antifade mountant (P36935; Molecular Probes, Eugene, OR).

Techniques: Western Blot, Staining, Mutagenesis

Journal: bioRxiv

Article Title: Contrasting synaptic roles of MDGA1 and MDGA2

doi: 10.1101/2023.05.25.542333

Figure Lengend Snippet: A, B, Schematic of the epitope-tagged HA-MDGA1 (A) and Myc-MDGA2 (B). C, D, Representative immunoblot of HA-MDGA1 and Myc-MDGA2 expression in the mouse brain, respectively, from postnatal day 3 to postnatal day 130, using α-tubulin as a loading control. WT P14 sample included for antibody specificity control. E, F, Quantification of HA-MDGA1 / α-tubulin and Myc-MDGA2 / α-tubulin ratios, respectively, normalized to postnatal day 3 values in the KI mice. P14 (peak HA-MDGA1 and Myc-MDGA2 expression) samples from WT mice were used as a control of tag antibody specificity. G, H, Representative immunoblots of HA-MDGA1 and Myc-MDGA2, respectively, using α-tubulin as a loading control, across brain regions. n=3-4 mice/condition for all Western blot analyses. I, Representative HA- MDGA1 immunofluorescence staining in coronal (top panel) and sagittal (bottom panel) slices of HA-MDGA1 mice at P15. J, Representative confocal images of HA staining with DAPI in WT (top) and HA-MDGA1 (bottom) of P20 HA-MDGA1 KI mouse hippocampus. High magnification insets of area CA1 are shown to the right. K, Quantification of HA puncta from high magnification confocal images shown in J (p<0.001, n=8 mice/genotype). Bar graphs represent mean ± SEM. *, p<0.005; **, p<0.01; ***, p<0.001, one-way ANOVA (E, F), unpaired two-tailed Student’s T-Test (K). Scale bars: I, 1 mm. J, 200 µm, insets: 50 µm. “SR”, stratum radiatum; “SP”, stratum pyramidale; “SO”, stratum oriens.

Article Snippet: Slides were mounted with either Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, # H-1200) or ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology, # 8961S).

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Contrasting synaptic roles of MDGA1 and MDGA2

doi: 10.1101/2023.05.25.542333

Figure Lengend Snippet: A, Low magnification (left) and high magnification (right) representative confocal immunofluorescence images labeling DAPI, HA-MDGA1, and either excitatory (Homer1b/c, top panels) or inhibitory (Nlgn2, bottom panels) postsynaptic marker in P20 HA-MDGA1 mouse hippocampi. B, Quantification of the colocalization of high intensity HA- MDGA1 and synaptic marker puncta (within 5 μm of each other) in the SR. The number of colocalized HA puncta is higher in HA-MDGA1 than WT with Homer1b/c (p <0.0001), but not with Nlgn2 p = 0.2219). In HA-MDGA1 samples, the proportion of HA-MDGA1 puncta colocalized with Homer1b/c is higher than with Nlgn2 (p< 0.0003), whereas in WT samples it is not (p=0.9981). C, D, In the MDGA1-HA/Homer1b/c colocalization experiment, the number of HA puncta was higher in KI samples (p<0.0001, C), but the number of Homer1b/c was not different (p = 0.7481, D). E, F, in the HA-MDGA1/Nlgn2 colocalization experiment, the number of HA puncta was higher in KI samples (p=0.0004, E), but the number of Nlgn2 was not (p = 0.4332, F). G, Low magnification (left) and high magnification (right) representative confocal immunofluorescence images labeling DAPI, HA-MDGA1, and either excitatory (vGluT1, top panels) or inhibitory (vGAT, bottom panels) presynaptic marker in P20 HA-MDGA1 mouse hippocampi. H, Quantification of the colocalization of high intensity HA-MDGA1 and synaptic marker puncta (within 5 μm of each other) in the SR. The number of colocalized HA puncta is higher in HA-MDGA1 than WT (p = 0.0012 with vGluT1, p = 0.049 with vGAT). In neither HA- MDGA1 samples (p = 0.0958) nor in WT samples (p=0.9981) the proportion of HA-MDGA1 puncta colocalized with vGluT1 and vGAT are significantly different. I, J, In the MDGA1- HA/vGluT1 colocalization experiment, the number of HA puncta was higher in KI samples (p<0.0001, I), and the number of vGluT1 was not (p = 0.9033, J). K, L, in the HA- MDGA1/vGAT colocalization experiment, the number of HA puncta was higher in KI samples (p= 0.0029, K), but the number of vGAT was not (p= 0.1157, L). n = 5/8 mice/group. Bar graphs represent mean ± SEM. n.s., non-statistically significant; *, p < 0.05; **, p<0.01; ***, p<0.001, Two-way ANOVA followed Tukey’s post hoc test in B and H and unpaired T-test in C-F and I-L. Scale bars: Left panels: 200 µm. Right panels: 20 µm. “SR”, Stratum Radiatum.

Article Snippet: Slides were mounted with either Vectashield Antifade Mounting Medium with DAPI (Vector Laboratories, # H-1200) or ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology, # 8961S).

Techniques: Immunofluorescence, Labeling, Marker

Journal: International Journal of Oncology

Article Title: Simvastatin delays castration-resistant prostate cancer metastasis and androgen receptor antagonist resistance by regulating the expression of caveolin-1

doi: 10.3892/ijo.2019.4774

Figure Lengend Snippet: Simvastatin downregulates the expression of Cav-1 via cholesterol de novo synthesis in cells. (A-C) PC3 and En-R cells were treated with 10 µ M simvastatin for 24 h, 10 mM methyl-β-cyclodextrin for 1 h and 10 µ M cholesterol for 5 h. (A and B) Western blot analysis of the expression of Cav-1 in detergent-resistant lipid-rich membrane fractions. (C) Cav-1 was detected using an immunofluorescence assay; cell nuclei were stained with DAPI (magnification, ×200). Arrows mark the Cav-1 expression in membranes. (D and E) PC3 and En-R cells were treated with simvastatin at 0, 1, 10 and 20 µ M for 24 h. Western blot analysis was performed to detect the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and Cav-1. * P<0.05, ** P<0.01 and *** P<0.001. En-R, enzalutamide-resistant LNCaP cells; Cav-1, caveolin-1.

Article Snippet: Cell nuclei were stained with DAPI (ZLI-9557; ZSGB-BIO) at 37°C for 5 min. Immunofluorescence images were obtained upon incubation in 50% glycerol using a fluorescence microscope (Nikon Corp.).

Techniques: Expressing, Western Blot, Membrane, Immunofluorescence, Staining