Journal: The EMBO Journal

Article Title: Mapping protein interactions of sodium channel Na V 1.7 using epitope‐tagged gene‐targeted mice

doi: 10.15252/embj.201796692

Figure Lengend Snippet: Schematic illustrating the affinity purification (ss‐AP and TAP) procedure using the tandem affinity tags separated with a TEV cleavage site. The proteins from DRG and olfactory bulbs were extracted in 1% CHAPS lysis buffer. After single‐step and tandem affinity purification, TAP‐tagged Na V 1.7 was detected using Western blotting with anti‐HAT antibody. The proteins from different tissues including hypothalamus, sciatic nerve, spinal cord, olfactory bulbs and DRG from KI mice, and pooled tissues from WT mice were extracted in 1% CHAPS lysis buffer. After single‐step affinity purification, TAP‐tagged Na V 1.7 was detected using Western blotting with anti‐HAT antibody. The interaction between TAP‐tagged Na V 1.7 and identified Na V 1.7 protein–protein interactors including Scn3b, Syt2, Crmp2, Gprin1, Lat1 and Tmed10 was validated using a co‐immunoprecipitation in vitro system. The expression vectors containing cDNA of validated genes were cloned and transfected into a HEK293 cell line stably expressing TAP‐tagged Na V 1.7. After transfection, TAP‐tagged Na V 1.7 complexes were immunoprecipitated with anti‐FLAG antibody, and the selected candidates were detected with their specific antibody using Western blotting. The results showed the expected sizes of Scn3b (32 kDa), Syt2 (44 kDa), Crmp2 (70 kDa), Gprin1 (two isoforms: 80 kDa and 110 kDa), Lat1 (57 kDa) and Tmed10 (21 kDa). Tissue expression pattern of TAP‐tagged Na V 1.7. The proteins were extracted from different tissues in both KI and WT littermate control mice and anti‐FLAG used to detect TAP‐tagged Na V 1.7 using Western blotting. Anti β‐tubulin was used as a loading control. The validation of selected Na V 1.7 protein interactor candidates with Nav1.7 endogenous expressing DRG tissue. First, the proteins from DRG of TAP‐tagged Na V 1.7 mice were extracted in 1% CHAPS lysis buffer. Na V 1.7 complexes were then immunoprecipitated by anti‐FLAG M2 magnetic beads. Thirteen Na V 1.7 interactor candidates including Scn3b (32 kDa), Syt2 (44 kDa), Crmp2 (70 kDa), Gprin1 (110 kDa), Lat1 (57 kDa), Tmed10 (21 kDa), Akap12 (191 kDa), Nfasc (138 kDa), Ntm (38 kDa), Kif5b (110 kDa), Ank3 (243 kDa) and Pebp1 (23 kDa) were detected with their specific antibodies using Western blotting. Co‐immunoprecipitation of Na V 1.7 with Ca V 2.2. Left panel shows negative Western blot results for pull‐down of transiently transfected HA‐tagged Ca V 2.2 from TAP‐tagged Na V 1.7 complex (HAT antibody for detection) in TAP‐tagged Na V 1.7 HEK293 stable cell line. Right panel shows control blot from whole‐cell lysate of HA‐tagged Ca V 2.2 and TAP‐tagged Na V 1.7. Source data are available online for this figure.

Article Snippet: The following plasmids were obtained from Addgene (Cambridge, MA): Tmed10 (TMED‐BIO‐HIS, #51852), Lat1 (pEMS1229, #29115).

Techniques: Affinity Purification, Lysis, Western Blot, Immunoprecipitation, In Vitro, Expressing, Clone Assay, Transfection, Stable Transfection, Control, Biomarker Discovery, Magnetic Beads

Journal: The EMBO Journal

Article Title: Mapping protein interactions of sodium channel Na V 1.7 using epitope‐tagged gene‐targeted mice

doi: 10.15252/embj.201796692

Figure Lengend Snippet: Identified Na V 1.7‐associated proteins (only appearing in KI group + selected candidates)

Article Snippet: The following plasmids were obtained from Addgene (Cambridge, MA): Tmed10 (TMED‐BIO‐HIS, #51852), Lat1 (pEMS1229, #29115).

Techniques: Marker, Biomarker Discovery