Journal: Molecular & Cellular Proteomics : MCP

Article Title: Identification of Cathepsin L as the Molecular Target of Hydroxychloroquine With Chemical Proteomics

doi: 10.1016/j.mcpro.2025.101314

Figure Lengend Snippet: Validation and characterization of the physical interaction between HCQ and cathepsin L (CTSL). A , in-gel fluorescence scan of HEK293T cells transfected with indicated plasmids followed by HCQ or HCQ-P treatment. The gray dashed lines indicate bands corresponding to the expected molecular weights on the fluorescent gel and the overexpressed proteins on the Western blot. The fluorescent gel and the Western blot were derived from the same gel. B , thermal stability graph of endogenous CTSL analyzed using immunoblot with a CTSL antibody. Each point of the curve was calculated from three independent biological replicates. C , cartoon showing HCQ (as stick ) and its binding pocket in CTSL heavy chain. (LibDock score: 111.63). Docking result was shown according to the hydrophobicity level. D , 2D interaction diagram of HCQ with amino acid residues of CTSL docking cavity. A conventional hydrogen bond was noted between the hydroxyl group of HCQ and Ser117 of CTSL. E , fluorescence labeling of CTSL or CTSL mutant expressed in HEK293T cells treated with HCQ-P. HCQ, hydroxychloroquine; HCQ-P, photo-crosslinking probe of HCQ; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: The antibodies used were as follows: CTSL antibody (CST #71298), FLAG-tag antibody (CST #14793), β-actin antibody (AF7018; Affinity), 488-FITC antibody (Thermo; A-11090), and goat anti-rabbit horseradish peroxidase (AS014; ABclonal).

Techniques: Biomarker Discovery, Fluorescence, Transfection, Western Blot, Derivative Assay, Binding Assay, Labeling, Mutagenesis