malachite green assay kit  (Echelon Biosciences)

Journal: Frontiers in Oral Health

Article Title: Emerging oral Treponema membrane proteins disorder neutrophil phosphoinositide signaling via phosphatidylinositol-4-phosphate 5-kinase

doi: 10.3389/froh.2025.1568983

Figure Lengend Snippet: MspA and MspTL increase phosphatase activity, but not via PTEN or SHIP activity. (A–D) Mouse bone marrow neutrophils were isolated, treated with 100 nM of recombinant MspA or MspTL for 30 min, then stimulated with 1 µM fMLP for 1 min prior to assays. (A) Total phosphatase activity was measured in neutrophil whole cell lysates by malachite green assay. (B) PTEN phosphorylation (Ser380) was assessed by immunoblot as a measure of activation. Neutrophils untreated or stimulated with fMLP served as negative and positive (+fMLP) controls while β-actin was included as a loading control. Right shows the mean ± SEM of 6 independent experiments, left shows a representative blot. (C) PTEN and (D) SHIP1 were immunoprecipitated from neutrophil lysate and then used in malachite green assays. (E–G) Mouse bone marrow neutrophils were isolated and treated with (E) 2 μM SF1670 (PTEN inhibitor), (F) 25 μM 3AC (SHIP1 inhibitor), or (G) 10 μM AS1949490 (SHIP2 inhibitor) for 30 min at 37C, then treated with 100 nM of recombinant MspA or MspTL for 30 min. Total phosphatase activity was measured in whole cell lysate by malachite green assay. Graphs show mean ± SEM of at least 3 independent experiments, with dots representing biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by ANOVA, (A) F 5, 12 = 8.969; (B–D) n.s.; (E) F 5, 12 = 14.08, (F) F 5, 12 = 13.39, (G) F 5, 12 = 25.90.

Article Snippet: Free phosphate release was measured using a Malachite Green Assay Kit (#K-1500, Echelon Biosciences) according to manufacturer instructions.

Techniques: Activity Assay, Isolation, Recombinant, Malachite Green Assay, Western Blot, Activation Assay, Control, Immunoprecipitation

Journal: Plant Biotechnology Journal

Article Title: KRN5b regulates maize kernel row number through mediating phosphoinositol signalling

doi: 10.1111/pbi.14463

Figure Lengend Snippet: KRN5b is the candidate gene of qKRN5b that positively affects the kernel number. (a) Physical location of KRN5b on chromosome 5 of the reference genome (B73_RefGen_v4). (b) Sequence variations between NX531 and SIL8 in the KRN5b coding region and promoter. The black rectangle stands for the coding region, while white rectangle stands for the UTRs. (c) Nucleotide diversity (π maize , π landrace , and π teosinte ) around KRN5b (blue interval). (d and e) KRN (d) and the relative KRN5b expression levels in the developing ears (e) of the recombinant lines used for QTL mapping. Two near‐isogenic lines (NIL) derived from Rec3 and Rec4 with or without introgression of qKRN5b from SIL8 were set for comparison for KRN and gene expression. Error bars indicate the standard deviation. * indicates a significant difference at P < 0.05; ** indicates a significant difference at P < 0.01, *** indicates a significant difference at P < 0.001. (f, g) Luciferase expression levels driven by P NX531 and P SIL8 (~1.7 kb), with the minimal 35S promoter used as the positive control. Data are presented as the mean (LUC/REN) ± standard deviation. The significance of any differences was assessed by Student's t ‐test. (h) Subcellular localisation of KRN5b. From left to right: bright field image, KRN5b‐GFP, SYP43‐mCherry, and merged fields. Scale bars = 10 μm. (i) Alignment of the conserved 5‐phosphatase domains in AtCVP2, AtCVL1, and KRN5b. (j) Protein sequence variation in the endonuclease/exonuclease/phosphatase family domain (EEP domain) of KRN5b. (k) Substrates of KRN5b. Phosphatase activity was analysed using the malachite green assay, which was performed three times, each with three technical replicates. The KRN5b protein had a high affinity for PI(4,5)P 2 , but a lower affinity for PI(3,4,5)P 3 and Ins(1,4,5)P 3 . Using three phosphoinositide phosphate species as substrates, the phosphatase activity of KRN5b NX531 was greater than that of KRN5b SIL8 and KRN5b mut . The significance of any differences was assessed by Student's t ‐test. ** P < 0.01; *** P < 0.001. (l) Analysis of the 5‐phosphatase activity of KRN5b using different concentrations of the substrate PI(4,5)P 2 . The enzyme activity of KRN5b NX531 was higher than that of KRN5b SIL8 . Data are presented as the mean ± standard deviation. The P ‐value was estimated by Student's t ‐test.

Article Snippet: The release of phosphorus ions was monitored using a malachite green phosphatase assay kit (Echelon Biosciences Inc. Salt Lake City, USA) according to the manufacturer's protocol.

Techniques: Sequencing, Expressing, Recombinant, Derivative Assay, Comparison, Standard Deviation, Luciferase, Positive Control, Activity Assay, Malachite Green Assay