h89 tocris  (Tocris)

Journal: eLife

Article Title: A bidirectional switch in the Shank3 phosphorylation state biases synapses toward up- or downscaling

doi: 10.7554/elife.74277

Figure Lengend Snippet: Figure 4. Increased PP2A activity maintains tetrodotoxin (TTX)-induced Shank3 hypophosphorylation. (A) Diagram showing the potential roles of kinases and phosphatases in regulating activity-dependent Shank3 phosphorylation. (B) Representative Western blot showing the impacts of inhibiting CAMKII (KN62, KN93) or PKA (H89) on Shank3 phosphorylation at baseline and upon TTX treatment. (C) Quantification of S1615 phosphorylation in (B) (two-way ANOVA with post-hoc Tukey’s test: DMSO vs. KN62, p>0.9999, DMSO vs. KN93, p=0.8148, DMSO vs. H89, p=0.9112, DMSO vs. picrotoxin (PTX), *p=0.0406, PTX vs. PTX/KN62, **p=0.0040, PTX vs. PTX/KN93, ****p<0.0001, PTX vs. PTX/H89, ****p<0.0001, n = 5 biological replicates). Dashed line indicates the DMSO control. (D) Quantification of PP2A activity after 1 hr TTX treatment (Un, n = 5, TTX, n = 5; paired t-test: **p=0.0018). (E) Quantification of PP2A activity after 24 hr TTX treatment (Un, n = 7, TTX, n = 7; paired t-test: *p=0.0129). (F, G) Western blot analyses showing changes in S1615 phosphorylation after 1 hr (F) or 24 hr (G) TTX treatment, with inhibition of PP2A by okadaic acid (OKA, 50 nM) during the

Article Snippet: DOI: https://doi.org/10.7554/eLife.74277 19 of 31 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent pAAV- CMV- PI- EGFP- WPRE- bGH Gift from James M. Wilson Addgene# 105530; RRID:Addgene_105530 Commercial assay or kit Lipofectamine 2000 Thermo Fisher Scientific Cat# 11668- 027 Commercial assay or kit Gibson Assembly Master Mix New England Biolabs Cat# E2611S Commercial assay or kit Lambda protein phosphatase New England Biolabs Cat# P0753S Commercial assay or kit BCA Protein Assay Kit Thermo Fisher Scientific Cat# 23227 Commercial assay or kit Protein- G Magnetic Beads Thermo Fisher Scientific Cat# 88847 Commercial assay or kit SimplyBlue SafeStain Thermo Fisher Scientific Cat# LC6060 Commercial assay or kit PP2A Immunoprecipitation Phosphatase Assay Kit Millipore Cat# 17- 313 Commercial assay or kit Ni- NTA Superflow Agarose Beads QIAGEN Cat# 30410 Chemical compound, drug Tetrodotoxin Tocris Cat# 1069 Chemical compound, drug Bicuculline methobromide Tocris Cat# 0109 Chemical compound, drug Picrotoxin Sigma- Aldrich Cat# P1675 Chemical compound, drug Okadaic acid Santa Cruz Cat# sc- 3513 Chemical compound, drug Tautomycetin Tocris Cat# 2305 Chemical compound, drug Fostriecin Tocris Cat# 1840 Chemical compound, drug KN62 Tocris Cat# 1277 Chemical compound, drug KN93 Tocris Cat# 1278 Chemical compound, drug H89 Tocris Cat# 2910 Chemical compound, drug Sequencing- grade trypsin Promega Cat# V5111 Chemical compound, drug Tandem Mass Tag (TMT) 10plex Thermo Fisher Scientific Cat# 90110 Software, algorithm Image Lab Software Bio- Rad RRID:SCR_014210 https://www.bio-rad.com/en-us/product/imagelab-software?ID=KRE6P5E8Z&source_wt= imagelabsoftware_surl Software, algorithm ZEN Black Zeiss RRID:SCR_018163 https://www.zeiss.com Software, algorithm Metamorph Molecular Devices RRID:SCR_002368 http://www.moleculardevices.com/Products/Software/ Meta-Imaging-Series/MetaMorph.html Software, algorithm Fiji Fiji RRID:SCR_002285 http://fiji.sc Software, algorithm GraphPad Prism GraphPad RRID:SCR_002798 http://www.graphpad.com/ Software, algorithm IGOR pro Wavemetrics RRID:SCR_000325 https://www.wavemetrics. com/products/igorpro/igorpro.htm Software, algorithm Spectrum mill v.7.00.208 Agilent Technologies Software, algorithm R v 4.0 The R Foundation RRID:SCR_001905 https://www.R-project.org/ Continued Continued on next page Wu, Tatavarty, Jean Beltran, et al. eLife 2022;11:e74277.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Control, Inhibition

Journal: BMC Complementary and Alternative Medicine

Article Title: 1α, 25-dihydroxy Vitamin D3 containing fractions of Catharanthus roseus leaf aqueous extract inhibit preadipocyte differentiation and induce lipolysis in 3T3-L1 cells

doi: 10.1186/s12906-019-2754-7

Figure Lengend Snippet: CRACE reduced lipogenesis and induced lipolysis in mature 3T3-L1 adipocytes by increasing cAMP level and PKA activation. a . mRNA expression analysis of lipogenesis related genes in CRACE treated and untreated mature adipocytes. b . mRNA expression analysis of major players of TAG lipolysis in CRACE treated and untreated mature adipocytes. In panel a and b mature adipocytes received CRACE (500 μg/mL equivalent) for 4 days. Bars represent mean ± SEM. n = 3. * p < 0.05 for untreated vs. CRACE treated. c . Treatment with CRACE (500 μg/mL equivalent) for 3 h increased lipolysis in mature 3T3-L1 adipocytes as measured by glycerol release. Isoproterenol (10 μM) was used as positive control. CRACE also increased lipolysis in cells where basal lipolysis was normalised with 100 nM adenosine (A) and 1 U/mL of adenosine deaminase (AD) treatment. Bars represent mean ± SEM. n = 3. * indicates p < 0.05 for untreated vs. different treatments and # indicates p < 0.05 for A + AD vs. A + AD + different treatments. d . CRACE increased PLN1 phosphorylation and decreased Akt phosphorylation (S473). Mature 3T3-L1 adipocytes were treated with CRACE (500 μg/mL equivalent) for 24 h followed by western blot analysis. Bars represent mean ± SEM, n = 3. * p < 0.05 for untreated vs. CRACE treated adipocytes. e . CRACE (500 μg/mL equivalent) increased intracellular cAMP level in mature 3T3-L1 adipocytes. 0.5 mM IBMX was added in all the cells for cAMP sustenance. Bars represent mean ± SEM, n = 3. * p < 0.05 for untreated vs. treated adipocytes. f . CRACE stimulated lipolysis by PKA activation in mature 3T3-L1 adipocytes. Glycerol release induced in mature adipocytes by CRACE (500 μg/mL equivalent) or isoproterenol in presence or absence of PKA inhibitor H89 was measured. CRACE induced lipolysis was significantly reduced by H89. Bars represent mean ± SEM, n = 3. * p < 0.05 for ‘given treatments’ vs. ‘treatment + H89’ (i.e. Untreated vs H89; CRACE vs CRACE + H89; ISO + CRACE vs ISO + CRACE + H89). Unpaired t-test (for panel 4 a , 4 b , 4 d and 4 e ) and one-way ANOVA followed by Bonferroni post-test (for panel 4 c and 4 f ) was performed to evaluate the statistical significance

Article Snippet: To test the effect of PKA inhibition on CRACE induced lipolysis, mature adipocytes were serum starved for 3 h. The cells were then pre-treated with 20 μM PKA inhibitor H89 (Tocris 2910) for 1 h in lipolysis buffer, followed by 3 h treatment with 500 μg/mL equivalent CRACE or 10 μM isoproterenol individually or together in presence of 20 μM H89.

Techniques: Activation Assay, Expressing, Positive Control, Phospho-proteomics, Western Blot