Chromogenic substrate

Formalin-fixed (FF) bacteria (2–4 × 10⁸ CFU total) or outer membrane protein (OMPs) fractions (5 µg total) isolated using Sarkosyl as previously described [34 (link)] were spotted on a nitrocellulose filter using a slot-blot apparatus (Thermo Fisher Scientific). The membranes were blocked with 1% BSAin TBS/Tween 20 (TBS-T) and incubated with pooled sera aliquots (1:200 dilution) overnight at 4 °C. An anti-mouse IgG secondary AP-conjugated antibody (Southern Biotech, Birmingham, AL, USA) was used to detect immunoreactive dots with NBT/BCIP (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue Tetrazolium) chromogenic substrate (Bio-Rad, Hercules, CA, USA).

In this study, the A2058 and SKMEL28 cells were lysed with RIPA lysis buffer (Beyotime, Jiangsu, China) at 4 °C for 30 min after the cells were transfected, which contain protease inhibitors (Roche, Indianapolis, IN, USA), and the BCA kit (Beyotime) was used to measure the protein concentration. Ten lane-volumes of proteins were separated using 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (0.22 µm; Millipore, Billerica, MA, USA). The membranes blocked with 5% nonfat milk (LP0033; Oxoid, Basingstoke, Hampshire, UK) for 1 h at room temperature, and incubated the membrane with relevant antibodies overnight at 4 °C: against METTL7A (1:1000, GTX65969; Genetex, Irvine, CA, USA), THBS1 (1:1000, 18304-1-AP; Proteintech, Rosemont, IL, USA), p53 (1:1000, 250143; ZenBio, Durham, NC, USA), p21 (1:1000, 2947; CST, Danvers, MA, USA), Cyclin D1 (1:1000, CST, 55506) and GAPDH (1:1000, GTX100118; Genetex). All antibodies were diluted with 5% Bovine Serum Albumin (WXBC4157V; Vetec, Hohenhameln, Germany). After being washed, the membrane was incubated with secondary antibodies (goat anti-mouse HRP (1:3000, A0563; Beyotime) or goat anti-rabbit HRP (1:3000, A0516; Beyotime). Secondary antibodies are diluted with 5% nonfat milk. A chromogenic substrate (Bio-Rad, Hercules, CA, USA) such as ECL is used for visualization.