Western blot analysis of TMC1 on different lysates with Rabbit anti-TMC1 antibody (HA723291) at 1/2,000 dilution. Lane 1: 293T transfected with empty control cell lysate Lane 2: 293T transfected with TMC1 cell lysate Lysates/proteins at 15 µg/Lane. Predicted band size: 88 kDa Observed band size: 88 kDa Exposure time: 3 minutes; ECL: K1801; 4-20% SDS-PAGE gel. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723291) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

Immunocytochemistry analysis of 293T cells labeling TMC1 with Rabbit anti-TMC1 antibody (HA723291) at 1/2,000 dilution. 293T cells, transfected with empty control (top, negative) / Flag-tagged TMC1 (bottom, positive) expression vector, respectively, were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-TMC1 antibody (HA723291) at 1/2,000 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. Flag (HA601167, 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.