Journal: Biochemistry and biophysics reports

Article Title: Transmembrane 163 (TMEM163) protein interacts with specific mammalian SLC30 zinc efflux transporter family members.

doi: 10.1016/j.bbrep.2022.101362

Figure Lengend Snippet: Fig. 1. Co-immunoprecipitation assays of heterologously expressed TMEM163, ZNT1, ZNT2, ZNT3, and ZNT4 proteins. Co-expressions of TMEM163-DDK with ZNT1-HA, ZNT2-HA, and TMEM163-HA show heterodimerization as indicated by their respective eluted bands upon immunoblotting with A) anti-DDK monoclonal antibody (mAb) and B) anti-HA polyclonal antibody (pAb). Co-expressions of TMEM163-DDK with ZNT2-HA, and TMEM163-HA with ZNT4-DDK both show heterodimerization as indicated by their respective eluted bands upon immunoblotting with C) anti-DDK mAb and D) anti-HA pAb. Expression of TMEM163-HA or TMEM163-DDK in all trials serves as a positive (dimerization) control with itself, while single expressions of each respective protein serve as negative controls. L, protein ladder; I, input/cell lysate; E, elution; +, expressed; –, not expressed. Predicted molecular weight (MW) of human proteins: TMEM163 = 31.5 kDa, ZNT1 = 55.3–63.0 kDa, ZNT2 variant 2 = 35.0 kDa, ZNT3 = 41.8 kDa, and ZNT4 = 47.4 kDa. The images are representative of N ≥6 independent trials.

Article Snippet: Primary antibodies were purchased from the following sources: anti-HA rabbit polyclonal antibody (pAb; #NB600-345, Novus Biologicals, Englewood, CO; #F3165, Millipore-Sigma, St. Louis, MO), anti-DDK (M2 Flag) mouse monoclonal antibody (mAb; NBP2-43570, Novus Biologicals; #H6908, Millipore-Sigma), anti-TMEM163 rabbit pAb (#NBP1-06608, Novus Biologicals), anti-ZNT1 rabbit pAb (#PA5-77768, Thermo Scientific), anti-ZNT2 rabbit pAb (#PA5-99761, Thermo Scientific), antiZNT3 mouse mAb (#TA501498, Origene Technologies, Rockville, MD), and anti-ZNT4 rabbit pAb (#PA5-80028, Thermo Scientific).

Techniques: Immunoprecipitation, Western Blot, Expressing, Control, Molecular Weight, Variant Assay

Journal: Biochemistry and biophysics reports

Article Title: Transmembrane 163 (TMEM163) protein interacts with specific mammalian SLC30 zinc efflux transporter family members.

doi: 10.1016/j.bbrep.2022.101362

Figure Lengend Snippet: Fig. 2. Immunocytochemistry of TMEM163 heterologously co-expressed with ZNT proteins. Representative confocal images of cells co-transfected with TMEM163-DDK and: A) ZNT1-HA, B) ZNT2-HA, C) ZNT3-HA, and D) ZNT4-HA. The DDK peptide was detected by anti-DDK mAb and visualized with anti-mouse secondary antibody conjugated with Alexa-488 (green). The HA peptide was detected by anti-HA pAb and visualized with anti-rabbit secondary antibody conjugated with Alexa-568 (red). DAPI stains the nuclei blue. Arrow indicates co-localization of TMEM163 and ZNT protein heterodimers within their respective cellular sites. Arrowhead indicates non-overlapping subcellular localization of protein homodimers. Protein co-localization was semi-quantitatively determined using the pixel- based and object-based analyses pipeline of the Cell Profiler software. Pearson correlation coefficient (PCC) shows the degree of overlapping fluorescence be tween the two channels. The fluorescent objects (in percent) provide the number of overlapping objects between the two channels. Scale bar: 100 μm. (For inter pretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary antibodies were purchased from the following sources: anti-HA rabbit polyclonal antibody (pAb; #NB600-345, Novus Biologicals, Englewood, CO; #F3165, Millipore-Sigma, St. Louis, MO), anti-DDK (M2 Flag) mouse monoclonal antibody (mAb; NBP2-43570, Novus Biologicals; #H6908, Millipore-Sigma), anti-TMEM163 rabbit pAb (#NBP1-06608, Novus Biologicals), anti-ZNT1 rabbit pAb (#PA5-77768, Thermo Scientific), anti-ZNT2 rabbit pAb (#PA5-99761, Thermo Scientific), antiZNT3 mouse mAb (#TA501498, Origene Technologies, Rockville, MD), and anti-ZNT4 rabbit pAb (#PA5-80028, Thermo Scientific).

Techniques: Immunocytochemistry, Transfection, Software, Fluorescence

Journal: Biochemistry and biophysics reports

Article Title: Transmembrane 163 (TMEM163) protein interacts with specific mammalian SLC30 zinc efflux transporter family members.

doi: 10.1016/j.bbrep.2022.101362

Figure Lengend Snippet: Fig. 4. Semi-quantitative analysis of cell surface expression of TMEM163 and ZNT proteins. WB of TMEM163-DDK co-expressed with each of ZNT1-HA, ZNT2-HA, ZNT3-HA, and ZNT4-HA proteins probed with anti-DDK to detect TMEM163 (A) and anti-HA peptide to detect each ZNT protein (B). The WB images associated with the analysis are shown in Fig. S6. Protein band in tensities were analyzed using Image J. The numerical values were semi- quantitatively measured using the area under the curve (AUC) tool. The AUC values from proteins that appear as doublet were averaged. Lysate: Input; PM: plasma membrane; A.U. = arbitrary unit.

Article Snippet: Primary antibodies were purchased from the following sources: anti-HA rabbit polyclonal antibody (pAb; #NB600-345, Novus Biologicals, Englewood, CO; #F3165, Millipore-Sigma, St. Louis, MO), anti-DDK (M2 Flag) mouse monoclonal antibody (mAb; NBP2-43570, Novus Biologicals; #H6908, Millipore-Sigma), anti-TMEM163 rabbit pAb (#NBP1-06608, Novus Biologicals), anti-ZNT1 rabbit pAb (#PA5-77768, Thermo Scientific), anti-ZNT2 rabbit pAb (#PA5-99761, Thermo Scientific), antiZNT3 mouse mAb (#TA501498, Origene Technologies, Rockville, MD), and anti-ZNT4 rabbit pAb (#PA5-80028, Thermo Scientific).

Techniques: Expressing, Clinical Proteomics, Membrane