Horseradish peroxidase

The anti-LPH polyclonal antibody was generated using female New Zealand white rabbits, as female is sensitive with lower doses of antigen and have higher response to immunization than male⁵⁶ . Female New Zealand white rabbits (6-8 weeks, 2 kg body weight) were immunized through repeated intradermal injections of LPH (1:1 emulsified in Freund’s adjuvant). After the final boost, blood was collected and serum was prepared. The polyclonal antibody was purified using Pierce™ Protein A IgG Purification Kit (Thermo Fisher Scientific). Immunoblot was performed to detect the presence of LPH in the culture supernatant of L. casei, L. rhamnosus, and L. paracasei. Probiotics were grown in Man, Rogosa, and Sharpe medium at 37 °C, 24 h without agitation. The cell lysates and culture supernatants were harvested, separated on SDS-polyacrylamide gel, and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5 % skim milk and incubated with rabbit polyclonal LPH antibody (1:5000) overnight. Expression of LPH was detected using goat anti-rabbit IgG antibody conjugated with horseradish peroxidase (1:5000, Proteintech, catalog number SA00001-2) and enhanced chemiluminescence reagent kit. The uncropped scans of immunoblots were provided in the Source Data file.

Cells were collected, and the protein was extracted with RIPA lysate. Then the protein was denatured by boiling at 99°C for 5 min, and quantified with BCA Protein Assay Kit (Beyotime Biotechnology Co, Jiangsu, China). After SDS-PAGE electrophoresis and transmembrane, the protein was binded to PVDF membrane, then blocked the nonspecific sites with 5% skimmed milk powder. Meanwhile, a primary antibody, including CREB1 (dilution of 1:1000, Cell signaling Technology, USA), p-CREB1 (dilution of 1:1000, Cell signaling Technology, USA), VASP (dilution of 1:1000, Cell signaling Technology, USA), HA (dilution of 1:1000, Cell signaling Technology, USA), GAPDH (dilution of 1:5000, Proteintech, USA), should be prepared, and then incubating the PVDF membrane overnight at 4°C. The membrane was incubated with the corresponding secondary antibody horseradish peroxidase (dilution of 1:1000, Proteintech, USA) for 1 h at room temperature, and finally detected by ECL reagents (Tanon, Shanghai, China). The optical density of bands was measured by a computer-assisted imaging analysis system (Tanon, Shanghai, China) and the relative protein expression levels were normalized to GAPDH.

The cells were washed in PBS and lysed with RIPA buffer (Vazyme, China) supplemented with protease inhibitor cocktail (Roche, Germany). After quantification using One DropTM 1000+ (ANTPEDIA, China), proteins were separated by SDS-PAGE under denaturing conditions and transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% fat-free milk for 2 h at room temperature and incubated with primary antibodies for Gapdh (Proteintech, 60004-1-Ig, 1:5,000), Mll1 (CST, D668N, 1:1,000), Mll2 (CST, E6A8V, 1:1,000), Pax6 (Abcam, ab5790, 1:500), and H2b (Abcam, ab52599, 1:10,000). The membranes were then incubated with secondary antibodies conjugated with horseradish peroxidase (Proteintech, USA). Protein signals were visualized using the ECL detection reagent (Thermo Scientific) on UVP (Analytik Jena AG, Germany).