Fig. S1. High K+ increases intracellular calcium level.
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1 Fig. S1. High K + increases intracellular calcium level. (A) Neuronal activation measured by calcium imaging using Fura-2. Intracellular calcium levels were continuously monitored by the fura-2 florescence ratios (34 nm /38 nm) 2 sec before and 4 sec after 15 mm KCl wash-in. (B) The average levels of intracellular calcium (1 sec) before (blue line in A) and after (red line in A) the application of. Data are presented as mean ± s.e.m. and compared by paired t-test (n=2 cells). : p <.5, : p <.1. Fig. S2. The effect of endogenously released BDNF induced by high K + or TBS on activity-dependent sustained phosphorylation. (A) Cultured neurons were incubated with or without after (one hour) treatment. Proteins were collected at different time points (from to 4 h). Representative western blots are present. High K + alone fails to induce visible phosphorylation. (B) -Fc was added to cultures 3min before stimulation by for 1 hour and washed out, followed by 1nM BDNF treatment. Western blots and quantitative plots show that removal of endogenous BDNF by -Fc did not alter sustained activation. (C) was added to cultures 3 min before stimulation by or (one hour). Then the neurons were treated by 1 nm BDNF and collected at 4h. Representative Western blots and quantitative plots are presented. (D) Cultured neurons were incubated with or without after TBS application. Proteins were collected at different time points (from to 4 h). Representative western blots are present. TBS alone fails to induce visible phosphorylation. (E) and -Fc was added to cultures 3min before stimulation by TBS for 1 hour and washed out, followed by 1nM BDNF treatment. Western blots and quantitative plots show that blocking endogenous BDNF by -Fc did not inhibit but attenuated sustained activation. For (B), (C) and (E), data from 4 independent experiments (N = 4) were averaged and plotted. Data are presented as mean ± s.e.m. : p <.5, : p <.1, : p <.1; ANOVA Fig. S3. High K + converts transient to sustained CREB activation induced by BDNF. Representative Western blots (A) and quantitative plots (B) showing the time courses of BDNF-induced CREB activation in the presence of or 15 mm KCl. Data from 4 independent experiments (N = 4) were averaged and plotted.
2 Data are presented as mean ± s.e.m. and compared by paired t-test. : p <.5, : p <.1. Fig. S4. A few control experiments. (A-C) Specific blockade of CaMKII by AIP reverses the sustained activation. Hippocampal neurons were treated with the CaMKII inhibitors AIP peptides for 3 h or KN-93 for 3 min before stimulation by 15 mm KCl or (one hour). Then was added on the cultured neurons and collected at different time points (2h and 4h). Representative Western blots (A) and quantitative plots of 2-hour time point (B) and 4-hour time point (C) are presented. Note: at 2 or 4-hour time point, level in each condition was compared to DMSO+ + group, respectively. : p <.5, : p <.1; ANOVA. (D, E) The effect of on surface expression. Hippocampal neurons were treated with or for one hour followed by application. Cell surface proteins were biotinylated at h and 4h after BDNF application and collected using immobilized Streptavidin. Equal amounts of biotinylated proteins were loaded to analyze surface expression. Representative Western blots (D) and quantitative plots (E) are presented. Data from 4 independent experiments (N = 4) were averaged and plotted. Note: surface expression level in each condition was compared to time point of or KCl respectively. : p <.1; ANOVA.
3 Fig. S1 A.7 15mM KCl Fura-2 Ratio (34/38 nm) Baseline 15mM KCl (s) B Fura-2 Ratio (34/38 nm) Baseline 15mM KCl
4 (normalized to total ) (normalized to total ) (normalized to total ) Fig. S2 A Time (h) 1h no BDNF B PBS -Fc + 4h BDNF.4.2 PBS -Fc C D Time (h) Vehicle 1nM BDNF 4h PBS TBS Vehicle + BDNF + BDNF TBS + 4h E -Fc TBS + 4h Fc
5 pcreb (normalized to total CREB) Fig. S3 A B Time (h) pcreb CREB Time (h)
6 Fig. S4 A B AIP KN h 4 h 2 h 4 h 2 h 4 h 2 h 4 h 2 h DMSO AIP KN-93 + DMSO C h DMSO AIP KN-93 + D Surface Total E Surface /Total h 4h PBS 4h BDNF h 4h PBS 4h BDNF
7 Tabble S1: One-way ANOVA for Fig. 3. is performed with software GraphPad Prism, a post hoc test is done to show the individual differences: 15 mm NaCL + + Number of primary dendrites 6.47± ± ± ± ± ±.23 Number of branch points 1.64± ± ± ± ± ±.17 Column A B C D E F Number of primary dendrites: Bonferroni's Multiple Comparison Test Mean Diff. t P value 95% CI of diff Column A vs Column B P < to Column A vs Column C P > to.7156 Column A vs Column D P > to.5417 Column A vs Column E P > to.8842 Column A vs Column F P > to.625 Column B vs Column C P < to 1.95 Column B vs Column D P > to Column B vs Column E P < to Column B vs Column F P > to 1.86 Column C vs Column D P > to.2956 Column C vs Column E P > to Column C vs Column F P > to.8661 Column D vs Column E P > to Column D vs Column F P > to Column E vs Column F P > to.755 Number of branching points: Bonferroni's Multiple Comparison Test Mean Diff. t P value 95% CI of diff Column A vs Column B P < to Column A vs Column C P > to.3424 Column A vs Column D P < to Column A vs Column E P > to.235 Column A vs Column F P < to Column B vs Column C P > to 1.13 Column B vs Column D P > to.581 Column B vs Column E P > to.991 Column B vs Column F P < to Column C vs Column D P > to.8244 Column C vs Column E P > to.4924 Column C vs Column F P < to Column D vs Column E P > to 1.5 Column D vs Column F P < to Column E vs Column F P < to
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Supplementary Information for. Inhibitors of Hedgehog Acyltransferase Block Sonic Hedgehog Signaling. Marilyn D. Resh 1,2,6*
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Figure S1. Loss of Ena/VASP proteins inhibits filopodia and neuritogenesis. (a) Bar graph of filopodia number per stage 1 control and mmvvee (Mena/ VASP/EVL-null) neurons at 40hrs in culture. Loss of all
Measurement of diastolic and systolic calcium concentration assessed by Fura-2 dye
SUPPLEMENTARY MATERIALS HDAC inhibition improves the sarcoendoplasmic reticulum Ca 2+ -ATPase activity in cardiac myocytes Viviana Meraviglia, PhD 1, Leonardo Bocchi, PhD2, Roberta Sacchetto, PhD 3, Maria
Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.
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Supplementary fig. 1. Crystals induce necroptosis does not involve caspases, TNF receptor or NLRP3. A. Mouse tubular epithelial cells were pretreated
Supplementary fig. 1. Crystals induce necroptosis does not involve caspases, TNF receptor or NLRP3. A. Mouse tubular epithelial cells were pretreated with zvad-fmk (10µM) and exposed to calcium oxalate
SensoLyte 520 Cathepsin K Assay Kit *Fluorimetric*
SensoLyte 520 Cathepsin K Assay Kit *Fluorimetric* Catalog # 72171 Kit Size 100 Assays (96-well plate) Optimized Performance: This kit is optimized to detect Cathepsin K activity. Enhanced Value: Ample
Nature Methods: doi: /nmeth.4257
Supplementary Figure 1 Screen for polypeptides that affect cellular actin filaments. (a) Table summarizing results from all polypeptides tested. Source shows organism, gene, and amino acid numbers used.
Supporting Online Material for
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doi: 1.138/nature775 4 O.D. (595-655) 3 1 -ζ no antibody isotype ctrl Plated Soluble 1F6 397 7H11 Supplementary Figure 1 Soluble and plated anti- Abs induce -! signalling. B3Z cells stably expressing!