Ponceau s

Cat sera were diluted with PBS (1:1) and incubated at 95°C for 5 min. The PVDF membrane was soaked using methanol and placed on soaked Whatman filter paper. Three μl of diluted serum was dropped onto the PVDF (Bio-Rad, Hercules, CA, USA) and dried. Then, it was blocked with a blocking buffer for 30 min at room temperature. After that, the PVDF was incubated with a primary mouse monoclonal anti-Bcl-2 antibody (BioLegend, San Diego, CA, USA) (1:1,000 dilution) within a blocking buffer at room temperature for 2 h. It was then incubated with HRP secondary goat anti-mouse IgG (BioLegend) (1:2,000 dilution) in a blocking buffer for 45 min at room temperature. Finally, the membrane was stained with a DAB substrate and dried at room temperature. Another PVDF membrane was prepared for the purposes of determining the total protein in each serum. This PVDF has been stained with Ponceau S (Sigma-Aldrich, St. Louis, MO, USA) staining for 10 min at room temperature. The Image Studio Lite^TM program (LI-COR, Lincoln, NE, USA) was used for the purposes of quantifying the densitometry of proteins, which were in the ratio of Bcl-2/Ponceau S (12 (link), 13 (link)). Each typical dot blot was converted to grayscale before protein densitometry was quantified.

Cells were extracted in NP-40 lysis buffer (1% NP-40, 20 mM Tris, pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA), containing protease and phosphatase inhibitor (Thermo Fisher Scientific). Extracts separated by SDS-PAGE were transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked in TBS 5% milk and probed with primary antibodies (1 h to overnight), washed 4 × 5 min in TBS 0.1% Tween-20 before incubating with secondary antibodies conjugated to AlexaFluor680 or IRDye800 for 1 h, washed and read on Odyssey CLx imaging system (LI-COR). Proteins were quantified using ImageStudiolite software. In some experiments, equal protein loading was controlled for by staining the membrane after transfer using either Ponceau-S or Revert total protein stain solution (LI-COR). The quantified Revert signal from a whole lane was used for normalization. Antibodies used for Western blotting are listed in Table S2.