Gel imaging analysis system

Three iron transport-related proteins (DMT-1, TfR-1, and FP1) were detected by Western blotting. Frozen heart tissue samples were homogenized with 400 μL lysis buffer/20 mg tissue and then centrifuged at 12,000 r/min for 10 min, after which the supernatant was collected. Total proteins were loaded and separated on a 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel and then transferred to a nitrocellulose membrane. Blocking was performed with 5% skimmed milk powder in phosphate buffer saline (PBS), and then the membranes were incubated with primary antibodies overnight at 4°C. The membrane was washed three times (10 min each) to remove uncombined primary antibodies, and then it was incubated with secondary antibodies at room temperature for 90 min. After the membrane was washed three times to remove unbound secondary antibodies, the membrane was exposed and scanned by the Gel imaging analysis system (UVP, USA), and the gray value was automatically measured by the system. The primary antibodies included anti-DMT1 antibody (ab55735, Abcam), anti-transferrin receptor antibody [MEM-189] (ab1086, Abcam), and anti-ferroportin/SLC40A1 antibody (ab58695, Abcam).

Of note, 3-year-old ginseng seedlings (mean height 10.23 cm and 3 branches) were used for testing. A total of 45 strains of rust rot pathogens were isolated from different regions, such as Liaoning Province, Jilin Province, and Heilongjiang Province (Table 1). The tested medium used was potato dextrose agar (PDA) (200 g potato, 17 g agar, 20 g glucose, and 1,000 ml distilled water). The reagents and instruments include a new plant genomic DNA extraction kit (Tiangen, Beijing), DNA marker-C (Shenggong, Shanghai), 2 × Es Taq Master Mix (Dye) (Kangwei, Beijing), general electrophoresis instrument (ABI, USA), gel imaging analysis system (UVP, USA), biomicroscopy (NIKON, Beijing), acetic acid-sodium acetate buffer (50 mmol/L, pH 5.5), citric acid-sodium citrate buffer (50 mmol/L, pH 5.0), and 0.1 mol Na-acetate buffer (pH 5.0, containing 1 mol·L⁻¹ NaCl and 1 mmol·L⁻¹ EDTA). The universal primers used were ITS1 (5′-TCCGTAGGTGAAC-CTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATAT-GC-3′) [Shenggong, Shanghai PCR instrument (ABI, USA)].