All lanes: Western blot analysis of TLR2 with anti-TLR2 antibody [JM22-41] (ET1705-92) at 1/500 dilution. Lane 1: Wild-type THP-1 whole cell lysate. Lane 2: TLR2 knockout THP-1 whole cell lysate. ET1705-92 was shown to specifically react with TLR2 in wild-type THP-1 cells. No band was observed when TLR2 knockout samples were tested. Wild-type and TLR2 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-TLR2 antibody (ET1705-92, 1/500) and Anti-β-actin antibody (R1207-1, 1/1,000) 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).

Immunocytochemistry analysis of THP-1 cells labeling TLR2 with Rabbit anti-TLR2 antibody (ET1705-92) at 1/100 dilution. Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-TLR2 antibody (ET1705-92) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI. Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.