Kgp701

A total of 5 × 10⁶ MDA-MB-231 cells were seeded into 6-well plates and incubated overnight. The cells were then treated for 12 h with RuDA-NPs (50 μM) or RuDA-NPs (50 μM) and irradiated with 808 nm laser (0.5 W, 10 min, 300 J cm⁻²) using PBS as the control group. After that, the cells were lysed in cell lysis buffer (KeyGEN BioTECH, KGP701), and the whole proteins were obtained after centrifugation at 8050 × g for 5 min at 4 °C. A pre-stained protein molecular weight Marker, 20–120 kD, KeyGEN BioTECH, KGM441) was used to determine the different blots. Equal amounts of proteins were then transferred to nitrocellulose filter membranes and blocked with nonfat milk (5%) in TBST buffer at room temperature for 2 h. Then, the membranes were incubated with primary antibodies (anti-β-actin 1:1000, anti-NRF2 1:500, anti-HO-1 1:10000, and anti-HSP70 1:1000), washed for three times with cold TBST buffer, and finally incubated with secondary antibodies (Goat Anti-rabbit IgG, IgG-HRP, KeyGEN BioTECH, KGAA35, 1:500). The blots were visualized via a chemiluminescence light-based detector (G:BOX chemiXR5, SYNGENE) and analyzed with Gel-Pro 32 software. Full image is provided in the Source Data file. Antibody information: Rabbit anti-β-actin (ABCAM ab68226), anti-NRF2 (ABCAM ab62352), anti-HO-1 (ABCAM ab68477), and anti-HSP70 (ABCAM ab181606).

The erythroblasts sorted from spleen were lysed with cell lysis buffer (KGP701, KeyGEN Biotech) containing freshly prepared protease inhibitor for 30 min on ice. The supernatant was collected after centrifugation for 15 min at 4 °C. The loading buffer was added to the supernatant and samples were denatured on a metal heater at 95 °C for 5 min. The protein was separated with 12% SDS-PAGE and transferred onto PVDF membrane (1620177, Bio-Rad, Hercules, CA, USA) with 100 V for 2 h. After blocking with 5% skim milk in Tris-buffered saline Tween (TBST) solution for 1.5 h at room temperature, the membrane was incubated with 14-3-3z (1:600, sc-1019, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (1:5000, ab181602, Abcam, Cambridge, UK) antibodies overnight at 4 °C. The membrane was washed with TBST 3 times and incubated with a secondary antibody (1:5000, ab205718, Abcam) for 1 h at room temperature. ECL reagent (WBKlS0100, Millipore, Boston, MA, USA) was used to show protein bands and ImageJ software was used for quantitative analysis of the intensity.