Vertical electrophoresis system

To detect whether the A6 peptide was successfully linked to HSA, coomassie brilliant blue staining was used to qualitatively determine the difference in the molecular weight of HSA after modification. SDS-PAGE was performed using a Bio-Rad vertical electrophoresis system at 80 V for 30 min, which was subsequently changed to 120 V for 50 min. After electrophoresis, the SDS-PAGE gel was washed 3 times with double-distilled water to remove the residual SDS from the gel surface. Then the gel was incubated with coomassie blue fast staining solution for 2 h at room temperature. Subsequently, bright and clear bands were obtained after being washed with double-distilled water to remove the background color.
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to quantitatively examine the modification of A6 peptide linked to HSA through SMCC. The freeze-dried samples were re-dissolved in TA50 (0.1% trifluoroacetic acid (TFA): acetonitrile = 1:1). The mass spectra of HSA modified by different degrees of A6 peptide was acquired in a mass range between 30,000 Da and 200,000 Da.

The protein was extracted from the developing Zea mays seeds according to the protocol described in [45 (link)]. SDS-PAGE was performed using a Vertical Electrophoresis System (Bio-Rad, Shanghai, China). Proteins were separated in SDS-PAGE on 10% acrylamide gels. Anti-rabbit antibodies (ZmPHO1) were generated according to the protocol described in [28 (link)]. After electrophoresis, the proteins in polyacrylamide gels were transferred to nitrocellulose membranes using an Electrophoretic Transfer Cell (Bio-Rad). The transfer buffer contained 10% running buffer, 20% methanol, and 70% water. After this membrane was incubated in blocking buffer (5% solution of skim milk in 1% TBST) for one hour on a rotator and then incubated overnight at 4 °C. A 15 µL quantity of ZmPHO1 antibody (affinity pure antibody) was added with (Anti-PHO1) 1:1000 dilution in blocking buffer, then incubated for 1 h on rotator at room temperature. The gel membrane was washed three times for 10 min each in 1% TBST. HPR secondary binding antibody Rabbit IgG was added with 1:2000 dilution in blocking buffer. The gel membrane was incubated for one hour. The gel membrane was washed three times for 10 min each in 1% TBST. A resolving solution was added according to the company or manufacturer’s instruction to photograph the bands’ results.

We determined the expression of FTH1 and NRF2 proteins via Western blot analyses. Briefly, protein lysates were prepared from cultured cells or mouse tumor tissue using an ice-cold cell lysis buffer (ThermoFisher, United States) supplemented with a protease inhibitor cocktail (Roche, Switzerland). Equal amounts of proteins were separated by 12% SDS polyacrylamide gels using a vertical electrophoresis system (Bio-Rad, United States), and then proteins were transferred to PVDF membranes (Millipore, United States) using semi-dry transfer (Bio-Rad). After blocking with 5% BSA, the membranes were incubated with primary antibodies, including FTH1 (CST, 1:1000), NRF2 (Abcam, 1:1000) or GAPDH (Abcam, 1:10000), at 4°C overnight, followed by incubation with secondary antibodies for 1 hour at room temperature. Protein levels were detected using ChemiDoc XRS + Imagine System (Bio-Rad, United States), while data were analyzed using the Image Lab software.