All lanes: Western blot analysis of BTK with anti-BTK antibody [SN69-07] (ET1611-4) at 1/1,000 dilution. Lane 1: Wild-type U2932 whole cell lysate. Lane 2: BTK knockout U2932 whole cell lysate. ET1611-4 was shown to specifically react with BTK in wild-type U2932 cells. No band was observed when BTK knockout sample was tested. Wild-type and BTK knockout samples were subjected to SDS-PAGE. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM in TBST for 1 hour at room temperature. The primary Anti-BTK antibody (ET1611-4, 1/1,000) and Anti-Vinculin antibody (ET1705-94, 1/5,000) were used in 5% BSA at room temperature for 2 hours. Goat Anti-Rabbit IgG H&L (HRP) Secondary Antibody (HA1001) at 1:200,000 dilution was used for 1 hour at room temperature. Cell lysate was provided by Ubigene Biosciences (Ubigene Biosciences Co., Ltd., Guangzhou, China).

Immunohistochemical analysis of paraffin-embedded human tonsil tissue using anti-BTK antibody. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1611-4, 1/50) for 30 minutes at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.