Journal: bioRxiv

Article Title: Uncovering protein-protein interactions of the human sodium channel Nav1.7

doi: 10.1101/2023.04.18.537407

Figure Lengend Snippet: A . The gene expression levels of sodium channel alpha subunits and beta subunits in human DRG and HEK293 cells. B . The gene expression levels of Nav1.7 related-chaperonins in human DRG and HEK293 cells. C . The gene expression levels of kinesins in human DRG and HEK293 cells. D . The gene expression levels of Nav1.7 related-membrane anchoring proteins (ankyrins and contactins) in human DRG and HEK293 cells. E and F . The gene expression levels of Nav1.7-related post-transcriptional modification (phosphorylation, E and ubiquitination, F) proteins in human DRG and HEK293 cells.

Article Snippet: The membranes were blocked with 5% nonfat milk for 30 minutes before being treated with primary antibodies, including anti-FLAG (1:1000, Sigma, F1804), anti-Nav1.7 (1:500, Origene, TA329033), anti-CCT5 (1:1000, Origene, TA308298), and anti-TMED10 (1:1000, Origene, TA306375), overnight at 4°C.

Techniques: Expressing, Membrane, Modification

Journal: bioRxiv

Article Title: Uncovering protein-protein interactions of the human sodium channel Nav1.7

doi: 10.1101/2023.04.18.537407

Figure Lengend Snippet: A . Schematic diagram of TAP-tagged SCN9A cDNA construct. B . Immunohistochemistry with an anti-FLAG antibody was conducted on HEK293 cells stably expressing TAP-tagged hNav1.7 and parental HEK293 cells. Scale bar = 100 μm. C . Western-blot analysis for TAP-tagged hNav1.7 was performed in HEK293 cells stably expressing TAP-tagged hNav1.7 and parental HEK293 cells. D and E . Representative raw current traces of NaV1.7 from HEK293 cells stably expressing the TAP-tagged NaV1.7 and parental HEK293 cells in response to the activation pulse protocol shown. F and G . Representative raw current traces of NaV1.7 from HEK293 cells stably expressing the TAP-tagged NaV1.7 and parental HEK293 cells in response to the inactivation pulse protocol shown. H . The current-voltage relationship of the channel in HEK293 cells stably expressing the TAP-tagged hNav1.7 and parental HEK293 cells. I . The activation and inactivation voltage dependence of the channel in HEK293 cells stably expressing the TAP-tagged hNav1.7.

Article Snippet: The membranes were blocked with 5% nonfat milk for 30 minutes before being treated with primary antibodies, including anti-FLAG (1:1000, Sigma, F1804), anti-Nav1.7 (1:500, Origene, TA329033), anti-CCT5 (1:1000, Origene, TA308298), and anti-TMED10 (1:1000, Origene, TA306375), overnight at 4°C.

Techniques: Construct, Immunohistochemistry, Stable Transfection, Expressing, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: Uncovering protein-protein interactions of the human sodium channel Nav1.7

doi: 10.1101/2023.04.18.537407

Figure Lengend Snippet: A . Schematic illustrating the procedures involved in the affinity purification, SEC, and MS/MS of TAP-tagged hNav1.7. B . TAP-tagged NaV1.7 was detected using western blotting with anti-FLAG antibody after single-step and tandem affinity purification (SS-AP and TAP). Ci . A representative size exclusion chromatography of the protein sample. Cii . The resolved fractions isolated from size exclusion chromatography visualized using Coomassie blue staining. D . The validation of selected Nav1.7 protein interactor candidates Kif11, CCT5, and TMED10, where Nav1.7 complexes were immunoprecipitated with anti-FLAG M2 magnetic beads and then detected with their specific antibodies using Western blotting.

Article Snippet: The membranes were blocked with 5% nonfat milk for 30 minutes before being treated with primary antibodies, including anti-FLAG (1:1000, Sigma, F1804), anti-Nav1.7 (1:500, Origene, TA329033), anti-CCT5 (1:1000, Origene, TA308298), and anti-TMED10 (1:1000, Origene, TA306375), overnight at 4°C.

Techniques: Affinity Purification, Tandem Mass Spectroscopy, Western Blot, Size-exclusion Chromatography, Isolation, Staining, Immunoprecipitation, Magnetic Beads

Journal: bioRxiv

Article Title: Uncovering protein-protein interactions of the human sodium channel Nav1.7

doi: 10.1101/2023.04.18.537407

Figure Lengend Snippet: A . Expression of CCT5 in HEK293 cells in response to the transfection of CCT5 siRNA. n = 4, P < 0.05. B . Expression of TMED10 in HEK293 cells in response to the transfection of CCT5 siRNA. n = 4, P < 0.05. C-E . Representative raw current traces of NaV1.7 from HEK293 cells stably expressing the TAP-tagged NaV1.7 in response to the activation pulse protocol shown. Each trace shows a different condition. F . IV plot of Nav1.7 current density in the absence and presence of CCT5 siRNA. Compared with NaV1.7 basal currents (n = 12), CCT5 siRNA transfection significantly reduced the sodium channel density (n = 10, P < 0.01). G . IV plot of Nav1.7 current density in the absence and presence of TMED10 siRNA. Compared with NaV1.7 basal currents (n = 10), CCT5 siRNA transfection significantly reduced the sodium channel density (n = 10, P < 0.05).

Article Snippet: The membranes were blocked with 5% nonfat milk for 30 minutes before being treated with primary antibodies, including anti-FLAG (1:1000, Sigma, F1804), anti-Nav1.7 (1:500, Origene, TA329033), anti-CCT5 (1:1000, Origene, TA308298), and anti-TMED10 (1:1000, Origene, TA306375), overnight at 4°C.

Techniques: Expressing, Transfection, Stable Transfection, Activation Assay

Journal: Neurobiology of Pain

Article Title: Human-like cutaneous neuropathologies associated with a porcine model of peripheral neuritis: A translational platform for neuropathic pain

doi: 10.1016/j.ynpai.2018.07.002

Figure Lengend Snippet: Representative images of Cy3 immunofluorescent labeling (red) for Nav1.7 (left column), ETA (middle column), and ETB (right column) in two Sham28 (A–F) and two PNT28 (G–L) ipsilateral biopsies. Cell nuclei are counterstained with DAPI (blue fluorescence). E, epidermis; UD, upper dermis; SB, Stratum Basalis; SS, Stratum Spinosum; SG, Stratum Granulosum; SC, Stratum Corneum. Scale bar = 100 μm. A,D,G,J. Nav1.7 immunolableing in Sham28 biopsies was concentrated in SG and upper SS (brackets in A, D) and increased in intensity as well as expanding through the full depth of SS, encroaching on SB in PNT28 biopsies (brackets in G, J). Quantification was based on repeated sampling of average PI in the upper and lower keratinocytes demarcated by the broken and dotted line marquees, respectively. (B, E, H, K) ETA expression in Sham28 biopsies was concentrated in SB (brackets in B, E) and expanded through the full depth of SG and SS in PNT28 biopsies (brackets in H, K). Quantification was based on repeated sampling of average pixel intensities in the SG and SS and in SB demarcated by the broken and dotted line marquees, respectively. (C, F, I, L) ETB expression in Sham28 biopsies was concentrated in SG and upper SS (brackets in C, F) and diminished in PNT28 biopsies (brackets in I, L). Quantification was based on repeated sampling of average pixel intensities in the SG and SS demarcated by the broken line marquees. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Three additional slides from each biopsy were also prepared to detect keratinocyte immunolabeling with specific antibodies against: 2. voltage-gated sodium channel alpha subunit 1.7 ( Nav1.7 ; rabbit polyclonal, 1:500, Alomone).

Techniques: Labeling, Fluorescence, Sampling, Expressing

Journal: Neurobiology of Pain

Article Title: Human-like cutaneous neuropathologies associated with a porcine model of peripheral neuritis: A translational platform for neuropathic pain

doi: 10.1016/j.ynpai.2018.07.002

Figure Lengend Snippet: Keratinocyte average immunofluorescent pixel intensity (PI) from FC18, PC10, PC28, PNT28, and Sham28 for Nav1.7 (A), ETA (C), CGRP (E), and ETB (F). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005. (B) Ratio of lower to upper average PI for Nav1.7 (black and gray bars respectively). (D) Ratio of combined SG and SS to SB average PI for ETA (black and gray bars respectively).

Article Snippet: Three additional slides from each biopsy were also prepared to detect keratinocyte immunolabeling with specific antibodies against: 2. voltage-gated sodium channel alpha subunit 1.7 ( Nav1.7 ; rabbit polyclonal, 1:500, Alomone).

Techniques:

Journal: Neurobiology of Pain

Article Title: Human-like cutaneous neuropathologies associated with a porcine model of peripheral neuritis: A translational platform for neuropathic pain

doi: 10.1016/j.ynpai.2018.07.002

Figure Lengend Snippet: (A) Average keratinocyte Nav1.7 PI among upper and lower keratinocytes for ipsilateral biopsies (solid black and gray bars, respectively) and contralateral biopsies (open black and gray bars, respectively) from PNT1, PNT7, PNT14, and PNT 21 pigs (see , Part 2). (B) Ratios of lower divided by upper average keratinocyte Nav1.7 PI for ipsilateral and contralateral biopsies (solid and open bars, respectively). (A) and (B) show results from average ipsilateral Nav1.7 PI among upper and lower epidermal keratinocytes in PNT28 biopsies (solid black and gray bars, respectively) and ipsilateral Sham28 biopsies (open broken line black and gray bars, respectively), included from A and B.

Article Snippet: Three additional slides from each biopsy were also prepared to detect keratinocyte immunolabeling with specific antibodies against: 2. voltage-gated sodium channel alpha subunit 1.7 ( Nav1.7 ; rabbit polyclonal, 1:500, Alomone).

Techniques: