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Protein expression of soluble TNALP-Flag with single- and double-site homodimeric mutations. Purified WT or mutated TNALP-Flag using Flag-immunoprecipitation with Flag-peptide elution from six replicate transfection experiments. The elutions were pooled together and immunoblotted with ProteinSimple immunoblotting assay (Bio-Techne) with rabbit polyclonal anti-Flag antibody. Residual Flag-peptide in the elutions was used as a loading control. EV: empty vector control. TNALP, tissue-nonspecific alkaline phosphatase.

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: Protein expression of soluble TNALP-Flag with single- and double-site homodimeric mutations. Purified WT or mutated TNALP-Flag using Flag-immunoprecipitation with Flag-peptide elution from six replicate transfection experiments. The elutions were pooled together and immunoblotted with ProteinSimple immunoblotting assay (Bio-Techne) with rabbit polyclonal anti-Flag antibody. Residual Flag-peptide in the elutions was used as a loading control. 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: Expressing, Purification, Immunoprecipitation, Transfection, Western Blot, Control, Plasmid Preparation

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.

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