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

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

Journal: Cells

Article Title: Dissecting the Biological Functions of Various Isoforms of Ferredoxin Reductase for Cell Survival and DNA Damage Response

doi: 10.3390/cells15010062

Figure Lengend Snippet: Expression profiles of various FDXR isoforms. ( A ) Schematic presentation of FDXR isoforms that are generated from alternative splicing or usage of different promoters. P1 and P2 promoters induce expression of isoforms 1–6 and isoform 7, respectively. The gene ID for each of the isoforms is NM_024417.5 for isoform 1; NM_004110.6 for isoform 2; NM_001258012.4 for isoform 3; NM_001258013.4 for isoform 4; NM_001258014.4 for isoform 5; NM_001258015.3 for isoform 6; and NM_001258016.3 for isoform 7. ( B ) The protein structures of various FDXR isoforms. MLS, mitochondria localization signal. FAD, FAD-binding domain. NADP, NAPDH-binding domain. FDX1/2, FDX1/2-binding domain. ( C – E ) qRT-PCR was performed in triplicate to measure the transcript levels of various FDXR isoforms in HCT116 ( C ), MCF7 ( D ), and HepG2 ( E ) cells. Total FDXR expression was used to normalize each isoform expression. ( F ) MCF7 cells were transiently transfected with an empty pcDNA3 vector or a vector expressing C-terminal Flag-tagged FDXR isoforms 1, 4, and 7 for 24 h, followed by Western blot analysis with antibodies against Flag, FDXR, or actin. ( G ) Recombinant FDXR isoform 1 or 7 produced in BL21 was subjected to Western blot analyses using FDXR (left panel) or Flag (right panel) antibody. ( H ) MCF7 cells transfected with a control vector or a vector expressing Flag-tagged isoforms 1, 4, and 7, followed by immunofluorescence using Flag antibody. Mitochondria and nuclei were visualized with MitoTracker and DAPI, respectively. Scale bar: 20 μm.

Article Snippet: Rabbit polyclonal anti-FDXR antibody (catalog number: 15584-1-AP) and rabbit polyclonal anti-Flag antibody (catalog number: 20543-1-AP) were purchased from ProteinTech (Rosemont, IL, USA).

Techniques: Expressing, Generated, Alternative Splicing, Binding Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Recombinant, Produced, Control, Immunofluorescence