Journal: The Journal of Biological Chemistry
Article Title: N-linked glycosylation plays an essential role in the stability and function of tissue-nonspecific alkaline phosphatase
doi: 10.1016/j.jbc.2025.111092
Figure Lengend Snippet: Cellular retention of TNALP with N271Q single- and double-site homodimeric mutations. Detection of TNALP-Flag in cell lysates and in cell supernatant (extracellular) of N271Q mutants 16 h post-transfection and culture with 1 μM MG-132. The cells were lysed with RIPA buffer and briefly sonicated. The extracellular TNALP-Flag was purified from cell medium using Flag-immunoprecipitation with Flag-peptide. Immunoblotting was performed with Protein Simple immunoblotting assay (Bio-Techne) with mouse monoclonal anti-Flag antibody. Residual Flag-peptide in the elutions was used as a loading control for extracellular TNALP-Flag. Actin was used as a loading control for cell lysates with mouse monoclonal anti-actin antibody. The image is representative of one independent experiment. EV, empty vector control; TNALP, tissue-nonspecific alkaline phosphatase.
Article Snippet: Rabbit polyclonal anti-Flag (F7425, Merck, lot 0000120996) or mouse monoclonal anti-Flag M2 (F3165, Merck, lot 0000405712) antibody and mouse anti-actin monoclonal antibody (ACTN05, Invitrogen, lot WI3205973) were used for TNALP-Flag as well as Flag-peptide and actin detection, respectively.
Techniques: Transfection, Sonication, Purification, Immunoprecipitation, Western Blot, Control, Plasmid Preparation