Any kd mini protean tgx precast gel

Concentrated conditioned media samples were subjected to Western blot to visualize relevant protein markers. First, IP was performed on samples as described above. A Bradford assay was conducted to determine protein concentrations, and 5 μg of each sample was run on an AnykD Mini-PROTEAN TGX Pre-Cast Gel (Bio-Rad) at 135 V for 50 minutes. Protein was transferred to a PVDF membrane using a Bio-Rad Trans-Blot Turbo. Membranes were blocked in 5% skim milk for 1 hour at room temperature and incubated with the following primary antibodies overnight at 4°C: anti-HSP90 (37-9400; mouse monoclonal; Thermo Fisher Scientific), anti-CD63 (25682-1-AP; rabbit polyclonal; Thermo Fisher Scientific), anti-flotillin1 (ab133497; rabbit monoclonal; Abcam), anti-syntenin1 (ab19903; rabbit polyclonal; Abcam), and anti–histone H3 (ab1791; rabbit polyclonal; Abcam); these markers were selected based on the Minimal Information for Studies of Extracellular Vesicles 2018 guidelines (19 (link)) and a recent comprehensive overview of EV protein markers (53 (link)). Following primary antibody incubation, membranes were washed in 1× Tris-buffered saline with Tween 20 (TBS-T) and incubated with the corresponding IRDye 800CW goat anti-mouse or anti-rabbit secondary antibody (LI-COR Biosciences) for 1 hour at room temperature. Membranes were subsequently washed in 1× TBS-T and imaged on a LI-COR Odyssey CLx.

The dried culture supernatant was dissolved in sample buffer (8 M urea, 2%CHAPS, 50 mM dithiothreitol [DTT], 0.2% Bio‐Lyte 3/10 carrier ampholyte, 0.001%BPB, #1632108 Bio‐Rad Laboratories, Inc., Hercules, CA, USA) and adjusted to 0.4 mg/ml. A total of 125 μl (50 μg) of the prepared samples was applied to immobilized pH gradient (IPG) strips (ReadyStrip, pH 3‐10 non‐linear, # 1632002, Bio‐Rad Laboratories, Inc.), and isoelectric focusing (IEF) was performed using the PROTEAN® i12 IEF System (Bio‐Rad Laboratories, Inc.) according to the manufacturer's instructions. Later, the IPG strips were reduced with DTT, carbamidemethylated with iodoacetamide, applied to precast gels (Any kD Mini‐PROTEAN TGX Precast Gel # 4569031, Bio‐Rad Laboratories, Inc.), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was performed. The electrophoresed gels were stained with Flamingo Fluorescent Gel Stain (# 1610491, Bio‐Rad Laboratories, Inc.) or Coomassie Brilliant Blue (CBB) (Expedeon, Cambridge, UK). Image analyses of the stained gels were performed using a Gel Imaging system (Gel Doc EZ system, Bio‐Rad Laboratories, Inc.) and PDQuest 2‐D analysis software (Bio‐Rad Laboratories, Inc.).

Cells were washed with PBS and lysed in 1 X RIPA buffer with protease and Pierce EDTA free phosphatase inhibitors (Thermo Scientific, A32957), with the addition of 1 U/mL Benzonase nuclease (Millipore, 101697) to degrade genomic DNA. Proteins were separated by SDS-PAGE on AnykD mini -PROTEAN TGX precast gel (Biorad) and transferred to 0.45 μm nitrocellulose membranes (Cytiva, 10600041). Membranes were blocked for 1 hr at RT in LiCOR Odyssey blocking buffer (927–60001). Blots were incubated overnight at 4°C with the following primary antibodies: β-ACTIN (Abcam, 6276; RRID:AB_2223210, 1:5000), β-TUBULIN (Abcam, 179513, 1:5000) p-IRF3(S396) (Cell Signaling, 49475, 1:1000); IRF3 (Bethyl, A303-384A-M, 1:1000); ANTI-FLAG M2 (Sigma Aldrich, F3165; RRID:AB_259529, 1:5000); NWSHPQFEK (Genscript, A00626-40,1:5000); CGAS (Cell Signaling, 316595, 1:1000); SRP55 (Bethyl, A303-669A-M, 1:1000); VIPERIN (RSAD2) (EMD Millipore, MABF106, 1:1000); ATP5A1 (Bethyl, A304-940A-T, 1:1000); CYTOCHROME-C (Abcam, 133504, 1:1000). Membranes were washed 3 X for 5 minutes in PBS-Tween20 and incubated with appropriate secondary antibodies (LI-COR, 925–32210, 926–68071) for 1 hr at RT prior to imaging on a LiCOR Odyssey Fc Dual-Mode Imaging System.

Total protein was extracted from 100mg tissue of 3-week-old plants under non-treated conditions. The powder ground in liquid nitrogen was mixed with 200μl protein extraction buffer (50 mM Tris-HCl pH = 7.5, 150mM NaCl, 1mM EDTA, 10% glycerol, 1mM DTT, 1mM Pefablock SC (Roche), 1×Complete Protease Inhibitor Cocktail (Roche), 1% Triton X-100). The homogenized solution was incubated on ice for 30 min. Insoluble material was spinned down at 4°C for 10 min at 12000×g. The supernatant was collected to a new eppendorf tube and the protein concentration was determined by Bio-Rad assay.
Twenty μg of total protein was resolved by Any kD^™ Mini-PROTEAN^® TGX^™ Precast Gel (Bio-Rad) and transferred to nitrocellulose membranes (GE Healthcare) by semi-dry blotting. Blot was incubated in the primary antibody of PR2 (pathogenesis-related protein 2) (Agrisera) at a dilution of 1:2500 for 1h at room temperature with agitation, followed by washing with TBS-T for 3 times. Then the blot was incubated in the secondary antibody, HRP-conjugated Anti-rabbit IgG (Cell Signaling Technology) at a dilution of 1:5000 for 1h at room temperature with agitation. The membrane was washed again as above and developed with ECL prime western blotting detection reagent (GE Healthcare), and the signals were detected with ECL Hyperfilm (GE Healthcare) after 10 min of exposure.

1% of the IP samples were loaded onto an Any kD MINI-PROTEAN TGX precast gel (Bio-Rad, Hercules, CA) followed by standard procedures of immunoblotting. α-FLAG-M2-HRP (Sigma-Aldrich, St. Louis, MO) was used to detect FLAG-tagged proteins. 10% of the IP samples were separated on a 10% polyacrylamide gel before being subjected to silver staining compatible to mass spectrometry. Co-purifying proteins with FLAG-tagged ORMM1 were identified by tandem mass spectrometry using a nanoLC-LTQ-Orbitrap instrument, followed by database searching with MASCOT against TAIR10 [36 (link)].

The expression of FLAG-tagged Rad52 was analyzed by western blotting. Proteins were extracted as described previously (32 (link)). Twenty micrograms of proteins (2 μg/μl) were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis using Any kD Mini-PROTEAN TGX Precast Gel (Bio-Rad). Transfer to membrane was performed with iBind Western System (Thermo Fisher Scientific) according to the manufacturer's protocol. Primary and secondary antibodies to detect Rad52-FLAG were FLAG M2 mouse monoclonal antibody (1:1000, Sigma-Aldrich) and goat anti-mouse IgG-HRP (1:2000, Santa Cruz Biotechnology), respectively. Primary and secondary antibodies to detect α-tubulin (loading control) were anti-alpha Tubulin antibody [YOL1/34] (1:2000, GeneTex) and goat Anti-Rat IgG H&L (HRP) (1:2000, Abcam), respectively. Following incubation with Clarity Western ECL Substrate (Bio-Rad), chemiluminescent signals were detected with ChemiDocTouch system (Bio-Rad). Gel images were processed with ImageJ software. The process involved cropping and altering window-level settings.