Pork gelatin

For trT2-50 and actin co-staining in HUVECs, cells were cultured on PBS-covered slides in 12-well plates with 0.1% pork gelatin (Sigma-Aldrich). Cells were fixed with 3% Paraformaldehyde (PFA) (Merck Millipore, Dermstadt, Germany) containing 0.5% triton, washed three times with PBS and blocked with 5% donkey serum (Jackson ImmunoResearch). Rabbit anti-trT2-50 or mouse anti-hRNASET2 (Sigma-Aldrich) or rabbit anti-angiogenin (Merck Millipore) were added (1:100 dilution; prepared in 5% donkey serum) and incubated overnight at 4°C. After washing three times with TBST, the slides were incubated for 1 h with Alexa 488-conjugated anti- rabbit or anti- mouse antibodies (Invitrogen Life Technologies) or Rhodamine Red™-X (RRX)-conjugated anti- rabbit antibody (Jackson ImmunoResearch) and phalloidin tetramethylrhodamine B isothiocyanate–conjugated anti-rabbit antibody (Sigma-Aldrich). Then, the slides were washed and mounted with a mixture containing 30% mounting medium, 4′,6-diamidino-2- phenylindole (DAPI) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and 70% fluoromount (Sigma-Aldrich). The slides were viewed under a Leicactr4000 laser scanning confocal microscope.

Cells were cultured on PBS-covered slides in 12-well plates with 0.1% pork gelatin (Sigma-Aldrich) and were incubated with peptide K108-K133 or angiogenin. Cells were fixed with 3% Paraformaldehyde (PFA) (Merck Millipore, Dermstadt, Germany) containing 0.5% triton, washed three times with PBS and blocked with 5% donkey serum (Jackson ImmunoResearch). Rabbit anti-angiogenin (Merck Millipore) was added (1:100 dilution; prepared in 5% donkey serum) and incubated overnight at 4°C. After washing three times with TBST, the slides were incubated for 1 h with Alexa 488-conjugated anti-rabbit antibody (Invitrogen Life Technologies) and phalloidin tetramethylrhodamine B isothiocyanate–conjugated anti-rabbit antibody (Sigma-Aldrich), and then washed, and mounted with a mixture containing 30% mounting medium, 4′,6-diamidino-2-phenylindole (DAPI) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and 70% fluoromount (Sigma-Aldrich). The slides were viewed under a Leicactr4000 laser scanning confocal microscope.

LppZ-specific IgA levels in human cohorts were determined by homemade enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well polystyrene plates (Corning, NY, USA) were coated with 1 μg/mL purified LppZ protein diluted in bicarbonate/carbonate coating buffer (15 mM Na₂CO₃ and 35 mM NaHCO₃, pH 9.6) and incubated at 4°C overnight. After washing three times with PBST (PBS containing 0.05% Tween-20; Sangon Biotech), the wells were blocked with 200 μL/well buffer G (PBS containing 1% pork gelatin; Sigma) at 37°C for 1 h. Human plasma (100 μL/well, 1:100 diluted in buffer G) was added to the wells and incubated at 37°C for 1 h. After washing three times with PBST, the wells were incubated with HRP-conjugated goat-anti-human IgA (SouthernBiotech, AL, USA) working solution (100 μL/well, diluted in buffer G) at 37°C for 1 h. Tetramethylbenzidine (TMB) (BD Bioscience, CA, USA) solution (100 μL/well) was added and the plates were incubated at room temperature in the dark for 15 min. The reaction was stopped by adding 1 M H₂SO₄ (50 μL/well). The absorbance at 450 nm was detected within 5 min by PowerWaveXS2 microplate spectrophotometer (BioTek Instruments, Inc., VT, USA).