mruby2 sequence  (Addgene inc)

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: Inhibition of TORC1 reduces mating GPCR levels at the PM and represses the mating pathway. A - D , representative images of cells expressing ( A ) Ste2-mEnvy, ( B ) Ste3-mEnvy, ( C ) Gpr1-mEnvy, and ( D ) Pma1-mRuby2 treated with or without 0.2 μM rapamycin for 2 h. Scale bars represent 4 μm. E , quantification of Ste2-mEnvy, Ste3-mEnvy, Gpr1-mEnvy, and Pma1-mRuby2 mean fluorescent intensities at the plasma membrane (-Rap: Ste2 n = 212 cells, Ste3 n = 285 cells, Gpr1 n = 197 cells, Pma1 n = 270 cells. +Rap: Ste2 n = 277 cells, Ste3 n = , Gpr1 n = 214 cells, Pma1 n = ). Measurements were normalized to the mean of the untreated groups to produce percentages. Error bars represent ±SEM. ∗ = p < 0.05, ∗∗ = p < 0.01, and ∗∗∗ = p < 0.001. F , β-galactosidase (pFUS1-LacZ) mating transcription assays of cell cultures pretreated with or without 0.2 μM rapamycin for 2 h (n > 10 colonies). Error bars represent ±SEM. EC 50 and E max are reported with ±95% confidence intervals. G , quantitative mating assays in cells pretreated with or without 0.2 μM rapamycin for 2 h (n = 5 assays). Error bars represent ±SEM. ∗ = p < 0.05 by two-sample one-tailed unequal variance t test. GPCR, G-protein–coupled receptor; mEnvy, monomeric Envy; PM, plasma membrane; TORC, target of rapamycin complex.

Article Snippet: The sequence for PMA1 was amplified from BY4741 genomic DNA, and the mRUBY2 sequence was amplified from pFA6a-link-yomRuby2-Kan (Addgene Plasmid #44953) ( ).

Techniques: Inhibition, Expressing, Clinical Proteomics, Membrane, One-tailed Test

Journal: Nature Communications

Article Title: Design and evaluation of a tripartite chemogenetic fluorescent reporter for visualizing ternary protein complexes

doi: 10.1038/s41467-025-62241-8

Figure Lengend Snippet: a Constructs for the expression of bJun-pFAST 1-98 , bFos-pFAST 115-125 and H2B-pFAST 99-114 for probing the Fos·Jun·DNA ternary complex formation. b Representative confocal micrographs of HBR-2,5DM-treated HeLa cells co-expressing bJun-pFAST 1-98 , H2B-pFAST 99-114 and either bFos-pFAST 115-125 or bFosΔ(179-193)-pFAST 115-125 . Cells were treated with 10 μM HBR-2,5DM. Identical imaging settings were used for direct comparison. Scale bars 20 μm. Representative confocal micrographs of n > 100 cells from three independent experiments. c Relative nuclear tripartite-split-FAST fluorescence computed by normalizing the nuclear fluorescence intensity of tripartite-split-FAST I F (tripartite-split-FAST) with the fluorescence intensity I F (iRFP670) of co-expressed iRFP670. Each cell is color-coded according to the biological replicate it came from. The solid circles correspond to the mean of each biological replicate. The black line represents the mean ± SD of the three biological replicates. Source data are provided as a Source Data file.

Article Snippet: The plasmid pAG1713 allowing the mammalian expression of H2B-pFAST 99-114 -P2A-bJun-pFAST 1-98 -IRES-HA-mTurquoise2 was constructed by adding H2B sequence to pAG1256 (CMV pFAST 99-114 -P2A-bJun-pFAST 1-98 -IRES-mTurquoise2, which was itself constructed by adding bJun sequence from Addgene vector #22012 pBiFC-bJunVN173 to pAG1184) for expression of a fusion between H2B and pFAST 99–114 .

Techniques: Construct, Expressing, Imaging, Comparison, Fluorescence

Journal: bioRxiv

Article Title: High and low exogenous nitrate concentrations produce distinct calcium signatures in Arabidopsis roots

doi: 10.1101/2025.03.03.641058

Figure Lengend Snippet: Asynchronous normalized Ca 2+ signal responses of the root hair (RH) compared to non-root hair (NRH) bearing epidermal cells showing distinct signatures comprising waves and spikes under low (0.25 mM) and high (5 mM) NO 3 - treatment. (A) The kinetics of apparent Ca 2+ signal represented through the GFP fluorescence intensity responding to low NO 3 - treatment. The root tissue imaged shows the apparent Ca 2+ signal through GFP fluorescence with mRuby2 RFP background and captures the response 90 seconds after the NO 3 - treatment. The peak normalized Ca 2+ signal intensity was captured around 190 seconds for all RH sections (RH-base, RH-mid and RH-tip) while NRH cell had peak normalized Ca 2+ signal intensity was around 240 seconds after low NO 3 - treatment. (B) The kinetics of apparent Ca 2+ signal responding to high NO 3 - treatment for the selected root sections. The root tissue imaged shows the apparent Ca 2+ signal through GFP fluorescence with mRuby2 RFP background and captures the response 110 seconds after the NO 3 - treatment. The peak normalized Ca 2+ signal intensity was captured around 120 seconds for RH-tip, and close to 140 seconds for RH-mid and RH-base sections.

Article Snippet: Sequences encoding mRuby2 (#40260, ( ) and GCaMP6s (#40753, ( ) were obtained from Addgene (Watertown, MA).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: High and low exogenous nitrate concentrations produce distinct calcium signatures in Arabidopsis roots

doi: 10.1101/2025.03.03.641058

Figure Lengend Snippet: Asynchronous normalized Ca 2+ signal in the root sections in response to the low NO 3 - treatment. A representative root section for the low NO 3 - treated seedling is shown with the normalized Ca 2+ signal, where section S1 was distal to the root tip while S4 was proximal to the root tip. The root tissue imaged above shows the apparent Ca 2+ signal through GFP fluorescence with mRuby2 RFP background and captures the response 120 seconds after low NO 3 - treatment. The peak normalized Ca 2+ signal intensity was captured around 90 seconds for sections S1 and S2, while the peak intensity of the normalized Ca 2+ signal was close to 125 seconds for S3 and 60 seconds for S4.

Article Snippet: Sequences encoding mRuby2 (#40260, ( ) and GCaMP6s (#40753, ( ) were obtained from Addgene (Watertown, MA).

Techniques: Fluorescence