Boiled and unboiled wild-type (WT) and Smoothened homolog knockout (KO) T-47D cell extracts (30 μg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Smoothened homolog antibody [HL2975] (GTX640362) diluted at 1:1000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody, and the signal was developed with Trident ECL plus-Enhanced.

Boiled and unboiled SH-SY5Y whole cell and membrane extracts (30 μg) were separated by 5% SDS-PAGE, and the membrane was blotted with Smoothened homolog antibody [HL2975] (GTX640362) diluted at 1:1000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody. (WCE: whole cell extract; ME: membrane extract)

Boiled and unboiled mouse brain extract (50 μg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Smoothened homolog antibody [HL2975] (GTX640362) diluted at 1:1000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody, and the signal was developed with Trident femto Western HRP Substrate.

Non-transfected (–) and transfected (+) boiled and unboiled 293T whole cell extracts (30 μg) were separated by 7.5% SDS-PAGE, and the membrane was blotted with Smoothened homolog antibody [HL2975] (GTX640362) diluted at 1:5000. The HRP-conjugated anti-rabbit IgG antibody (GTX213110-01) was used to detect the primary antibody.