Mouse monoclonal actin antibody

Total protein from 2- and 3-DAP seeds of A619 and vks1 was extracted with radioimmunoprecipitation assay lysis buffer (Leagene, catalog number PS0013). Proteins were quantified by Bradford assays. Twenty micrograms of protein was separated by a 10% SDS-PAGE gel and then transferred electrophoretically to a PVDF membrane (Bio-Rad, catalog number 162-0177). The membrane was incubated with VKS1 antibodies at 4°C overnight and then incubated with the secondary antibody, goat anti-rabbit IgG-horseradish peroxidase (HRP; Abmart, catalog number M21002L). To detect the control protein ACTIN, a primary antibody, mouse monoclonal ACTIN antibody (Abmart, catalog number M20009L), and a secondary antibody, goat anti-mouse IgG-HRP (Abmart, catalog number M21001L), were used to perform the immune reaction. The dilution of antibodies was 1:1000. Finally, the membrane was treated with HRP chemiluminescent substrate reagent (Invitrogen, catalog number WP20005) and imaged using a Tanon-5200 system (Tanon).
To analyze the protein content of VKS1 in vks1-2, protein was extracted from the 2-DAP seeds of B73 and vks1-2.

Western blotting analyses were performed as described previously [47 (link)]. Briefly, a total amount of 30 μg proteins were extracted from the wounded leaf discs at various time points and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then total protein was electrotransferred onto polyvinylidene difluoride membranes. Immunoblotting was performed using JAZ1 antibodies against the Arabidopsis JAZ1 protein (Agrisera, http://www.agrisera.com/), then incubated with the secondary antibody goat anti-rabbit IgG-horseradish peroxidase (HRP) (Abmart, China). To detect the Actin, the primary antibody (mouse monoclonal Actin antibody) and the secondary antibody (goat anti-mouse IgG-HRP) were used to perform the immune reaction (Abmart, Shanghai, China). After incubation in the chemiluminescence detection solution LumiGLO (KPL, USA), membranes were imaged with a chemiluminescence image system Tanon 5500 (Tanon, Shanghai, China). Proteins were quantified with a BCA Protein Assay kit (Thermo, Shanghai, China).

A partial ARFTF17 protein fragment from the 480th to 644th amino acid was used by ABclonal (Wuhan, China) to make antibodies. To analyze accumulation of ARFTF17 protein in B73 and different fka1-1 mutants, total protein was extracted from the pericarp and endosperm at eight and 12 DAP with the non-zein buffer^{52 (link)}. Twenty μg of total protein was separated by 10% SDS-PAGE and transferred electrophoretically to a PVDF membrane. Immunodetection used ARFTF17 antibodies at a concentration of 1:1,000 at 4 °C overnight, followed by a secondary anti-rabbit-HRP at a concentration of 1:5,000 (Abmart, catalog number: M21002L). To detect the control protein, ACTIN, a primary antibody, mouse monoclonal ACTIN antibody (Abmart, catalog number: M20009L) and a secondary antibody, anti-mouse IgG-HRP (Abmart, catalog number M21001L), were used. To examine FLAG in fka1-1; ARFTF17pro:Flag-ARFTF17 plants, total protein was extracted from the pericarp. Immunoblotting used anti-FLAG (Sigma, A8592) as the primary antibody at a dilution of 1:1,000, and anti-mouse IgG-HRP (Abmart, M21001L) as the secondary antibody at a dilution of 1:5,000. The membranes were treated with a chemiluminescence substrate (Invitrogen, catalog number: WP20005), and the immunoreactive bands were detected using Tanon-5200 system.