alkaline phosphatase ap conjugate substrate kit  (Bio-Rad)

Journal: Plant Communications

Article Title: PHR1-like 7 and phosphatidic acid oppositely regulate TAG degradation and seed oil accumulation in Arabidopsis

doi: 10.1016/j.xplc.2025.101627

Figure Lengend Snippet: Detection of PHL7–PA interaction. (A) PHL7 production and purification. Recombinant PHL7 was expressed in E . coli . Ten micrograms of the supernatant from the cell lysate were separated on a 10% SDS–PAGE gel and analyzed by immunoblotting with an anti–6× His antibody (left) or by Coomassie Blue staining (right). (B) Protein–lipid overlay assay of PHL7 with different phospholipids. Mixed acyl species of PA, PC, PE, and PG were derived from chicken egg yolk. Chl, chloroform; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine. (C) Protein–lipid overlay assay testing PHL7 interaction with PA species. Ten micrograms of chicken egg yolk PA or synthetic PA species with defined acyl chains at the sn -1 and sn -2 positions were used. Protein purified from the empty vector (E.V.) served as a negative control. PHL7 was detected using an anti–6× His antibody conjugated to alkaline phosphatase. Experiments were repeated at least twice with similar results. Chl, chloroform; PA, phosphatidic acid. (D) Liposome-binding assay of PHL7 and PA. Liposomes were prepared with 0.6 μmol PA and 0.2 μmol PC. Ten micrograms of PHL7 protein were incubated with liposomes and detected by immunoblotting using an anti–6× His antibody conjugated to alkaline phosphatase. Liposomes containing 0.8 μmol PC without PA served as a negative control. One-tenth of the PHL7 protein input was used as a reference. Experiments were repeated at least twice with similar results.

Article Snippet: Proteins were detected using either alkaline phosphatase substrate (Bio-Rad, cat. #170-6432) or SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific, cat. #34577) according to the manufacturers’ instructions.

Techniques: Purification, Recombinant, SDS Page, Western Blot, Staining, Protein-lipid Overlay Assay (PLOA), Derivative Assay, Plasmid Preparation, Negative Control, Binding Assay, Liposomes, Incubation

Journal: Plant Communications

Article Title: PHR1-like 7 and phosphatidic acid oppositely regulate TAG degradation and seed oil accumulation in Arabidopsis

doi: 10.1016/j.xplc.2025.101627

Figure Lengend Snippet: Effect of PHL7 alterations on the expression of genes involved in TAG accumulation. (A) Transcript levels of PHL7 during seed development. Seeds collected at 6–14 days after flowering (DAF) were used for RNA extraction from WT, phl7-1 , and OE12 lines. TUB4 was used as the internal control. (B) Schematic representation of fatty acid biosynthesis, TAG accumulation, and TAG degradation during seed development. Key enzymes and proteins include WRINKLED 1 (WRI1), PA phosphatase (PAH), acyl-CoA:diacylglycerol acyltransferase (DGAT), phospholipid: diacylglycerol acyltransferase (PDAT), and TAG lipases (TGALs), including sugar-dependent 1 (SDP1), lipase 1 (LIP1), sugar-dependent 1-like (SDP1L), Myzus persicae –induced lipase 1 (MPL1), and senescence-associated gene 101 (SAG101). Monoacylglycerol lipases (MAGLs) include lysophospholipase 2 (LYSOPL2). (C) Effects of phl7-1 and PHL7 OE on the expression of genes involved in lipid biosynthesis and degradation in developing seeds at 10 and 12 DAF. Values are shown as means ± SD ( n = 3). Asterisks represent significant differences (∗∗ p < 0.01, ∗ p < 0.05; Student’s t- test).

Article Snippet: Proteins were detected using either alkaline phosphatase substrate (Bio-Rad, cat. #170-6432) or SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific, cat. #34577) according to the manufacturers’ instructions.

Techniques: Expressing, RNA Extraction, Control