Bis tris plus gels

Recombinant protein rSspA_P1/7, rSspB_ZY05719, rScpB_A909 (100 ng) were incubated with human or mouse complement component C5a (0.5 μg) (R&D) in HEPE++ buffer (20 mM HEPES, 140 mM NaCL, 5 mM CaCL₂, 2.5 mM MgCL₂) in a final volume 10 μL. At the same time, SS2 strain ZY05719 and S. agalactiae strain A909 in stationary phase (adjusted to 4 × 10⁶ CFU) were washed twice with PBS to incubated with human or mouse C5a (0.5 μg) (R&D) with THB in a final volume 10 μl. All samples were incubated at 37°C with 5% CO₂. After 16 h, samples were fractionated on 4–20% Bis-Tris plus gels (Genscript) and stained by Instant blue (Expedeon) for following SDS-PAGE analysis.

Western blotting was performed as we described before^{13 (link)}. Briefly, cells were washed with PBS and lysed. The concentration of total proteins was determined using a Nanodrop (Thermo Fisher, USA). Proteins were separated by 12% Bis–Tris Plus gels (Genscript, China), transferred to PVDF membrane (Millipore, USA), and blocked for 1 h at room temperature and incubated with primary antibodies, anti-transferrin receptor (TfR) (1:1000; ab214039, Abcam, USA), anti-NCOA4 (1:1000; A5695, ABclonal, China), anti-ferritin (1:1000; ab65080, Abcam), anti-ferroportin (FPN) (1:1000; ab78066, Abcam), anti-LC3B (1:1000; 83506, CST, USA), anti-HIF-1α (1:1000; ab2185, Abcam), anti-CDK9 (1:1000;2316,CST), anti-BRD4 (1:1000; ab128874, Abcam), anti-p38 (1:1000; 8690, CST), anti-Phospho-p38 (1:1000; 4511, CST), anti-JNK (1:1000; 9252, CST), anti-Phospho-JNK (1:1000; 4668, CST), anti-ERK1/2 (1:1000; 4695, CST), anti-Phospho-ERK1/2 (1:1000; AF1015, Affinity, China), or GAPDH (1:1000; MB001, Bioworld, USA). Subsequently, HRP-conjugated secondary anti-bodies including anti-rabbit and anti-mouse antibodies (Fcmacs, China) were incubated for 1 h. Blots were then visualized by a chemiluminescent imaging system (Tanon, China). The optical density of each lane was read using ImageJ (Bethesda, USA).