Horseradish peroxidase hrp linked anti rabbit secondary antibody

Single colony of V. vulnificus strain was inoculated into LB broth and cultured overnight at 37°C in a shaking incubator (200 rpm). The overnight cultures were diluted 200-fold with fresh LB medium and subsequently cultured at 37°C. The pellets were washed twice with DPBS (Welgene, Gyeongsan-si, Gyeongsangbuk-do, South Korea). Bacterial cells (2×10⁸ CFU) resuspended in SDS-PAGE sample buffer were boiled at 100°C for 10 min and separated by 10% SDS-PAGE gels before being transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% skim milk in Tris‐buffered saline containing 0.05% Tween 20 (TBS/T) for 2 h at room temperature and incubated with primary antibodies specific to TolCV1 (Guo et al., 2020 (link)), TolCV2 or RtxA1-D2 (Kim et al., 2013 (link)) at 4°C overnight. The membranes were rinsed with TBS/T for 1 h, incubated with horseradish peroxidase (HRP) linked anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h, and washed again with TBS/T for another 1 h. Protein bands were detected by the ECL Western blot analysis system (Advansta, Menlo Park, CA, USA). The intensity of bands was measured in arbitrary units (AU) by using ImageJ 1.50i software (National Institute of Health, USA).

Thyroid tissues were cultured in RPMI 1640 medium (WISENT, Nanjing, China, 350‐000‐CL) supplemented with PBS, spleen homogenate or 1 ng mL⁻¹ IGF‐1 (MedChemExpress, New Jersey, USA, HY‐P7070), and the inhibitor of the IGF‐1 receptor (picropodophyllin, AXL1717) (MEC, HY‐15494) for 6 h. Afterward, the tissues were subjected to Western blot analysis.
For the Western blot, tissue samples were lysed in RIPA buffer (Beyotime, P0013B) containing protease inhibitor PMSF (Beyotime, ST2573) at 4 °C for 30 min. The lysates were centrifuged at 12,000 rpm for 10 min, and the protein extracts were collected.
The protein extracts were separated by SDS‐PAGE electrophoresis and transferred to a PVDF membrane using the Mini‐PROTEAN Tetra Electrophoresis System (Bio‐Rad). The PVDF membrane was then incubated overnight at 4 °C with primary antibodies against Cyclin D1 (Abcam, ab134175, 1:10 000) or GAPDH (Proteintech, HRP‐60004, 1:10 000).
To detect the bound antibodies, a horseradish peroxidase (HRP)‐linked anti‐rabbit secondary antibody (Jackson ImmunoResearch, 111‐035‐003) was used. The HRP signal was visualized using 4200SF (Tanon Shanghai). This allowed for the visualization of Cyclin D1 and the internal control protein GAPDH on the PVDF membrane.