Journal: Molecular Pain

Article Title: Nonmuscle myosin II isoforms interact with sodium channel alpha subunits

doi: 10.1177/1744806918788638

Figure Lengend Snippet: Interaction of myh9 with Na α subunits expressed in the mouse brain tissues. WT type (In, lane 1 in (a) and (b)) or Na v 1.6 KO ( Scn8a medtg ; In, lane 1 in (c)) mouse brain tissue lysates were PC with mouse IgG2b isotypes (PC, lane 2 in (a), (b), and (c)) prior to IP using mouse anti-myh9 antibodies (IP, lane 3 in (a), (b), and (c)) of the IgG2b isotypes. Myh9 coimmunoprecipitated pan-Na v ((i) in (a)), Na x ((ii) in (a)), and Na v 1.2 ((iv) in (a)) subunits, but not Na v 1.1 subunits ((iii) in (a)), expressed in mouse brain tissues. Na v 1.6 subunits ((i) in (b)) were not coimmunoprecipitated with myh9 expressed in the mouse brain tissues. As expected, there was not any Na v 1.6 immunoreactivity from Scn8a medtg mouse brain tissues ((i) in (c)). Myh9 also coimmunoprecipitated β-actin ((v) in (a) and (ii) in (b) and (c)) and MRLCs ((iii) in (b) and (c)) expressed in mouse brain tissues. The MRLC immunoreactive signal from the mouse brain tissue lysates is barely detectable. Mouse IgG-HC (panel (ii) in (b) and (c)) and IgG-LC (panel (iii) in (b) and (c)), which are separated from their intact immunoglobulins (i.e., used for PC or IP) upon denaturation, could be seen as these blot sections are probed with mouse (anti-β-actin and anti-MRLC) antibodies. Additional information is available in Figure S1. IgG-LC: immunoglobulin light chain; IgG-HC; immunoglobulin heavy chain; In: lysate input; IP: immunoprecipitation; KO: knockout; mIgG2b: mouse immunoglobulin isotype 2b; MRLC: myosin regulatory light chain; myh: myosin heavy chain; Na v : voltage-sensitive sodium channel; Na x : nonvoltage-dependent sodium channel; PC: precleared; WT: wild type.

Article Snippet: Polyclonal antibodies against Na v 1.2 (1:200; Alomone), Na v 1.3 (1:200; Alomone), Na v 1.6 (PN4; 1:200; Sigma), Na v 1.7 (1:200; Alomone), Na v 1.8 (1:200; Alomone), Na v 1.9 (1:200; Alomone), Na x (1:1000; Abcam), β-II spectrin (1:2000; Abcam), β-III spectrin (1 µg/mL; Santa Cruz Biotechnology), and GFP (1:1000; Invitrogen) were also used for immunoblotting.

Techniques: Immunoprecipitation, Knock-Out

Journal: Molecular Pain

Article Title: Nonmuscle myosin II isoforms interact with sodium channel alpha subunits

doi: 10.1177/1744806918788638

Figure Lengend Snippet: Interaction of myh9 and myh10 with Na v subunits expressed in the rat brain tissues. (a) Myh9 and myh10 interact with pan-Na v subunits expressed in the rat brain tissues. Rat brain tissue lysates (In, lanes 1 and 9) were PC with mouse IgG2b isotypes (PC, lanes 2 and 8) and mouse IgG1 isotypes (PC, lane 6) prior to IP using indicated antibodies (lane 3 = myh9, lanes 4 and 10 = myh10, lane 5 = KIF5B, and lane 7 = AnkG) of the same isotypes as those were used for preclearing. IP complexes in the gel were loaded following the loading of their respective isotype (PC) complexes. Pan-Na v subunits (i) were coimmunoprecipitated with myh9, myh10, KIF5B, and AnkG expressed in the rat brain tissues. CoIP of pan-Na v subunits by AnkG and KIF5B served as positive controls. Myh9 and myh10 coimmunoprecipitated β-actin (ii) and MRLCs (iii) expressed in the rat brain tissues. Anti-MRLC antibodies poorly detect their antigens from rat brain tissue lysates (lane 1 in (iii)). AnkG also coimmunoprecipitated β-actin and MRLCs expressed in the rat brain tissues. Denatured mouse IgG-HC (iii) separated from their intact immunoglobulins (i.e., used for PC or IP) could be seen in the immunoblot, as this section of the blot was probed with mouse anti-β-actin antibodies. (b) Interaction of myh10 with Na α subunits expressed in the adult rat brains. Rat brain tissue lysates (In, lane 1) were PC with mouse IgG1 isotypes (PC, lane 2) and mouse IgG2b isotypes (PC, lane 4) prior to IP using antibodies for AnkG (IP, lane 3) and myh10 (IP, lane 5) of the of the same isotypes as those were used for preclearing. Loading of isotype (PC) complexes in the gel preceded those of the IP complexes. Myh10 coimmunoprecipitated Na x (i) and Na v 1.2 (iii) subunits, but not Na v 1.1 (ii) and Na v 1.6 (iv) subunits, from rat brain tissues. As expected, AnkG coimmunoprecipitated Na v 1.1 (ii), Na v 1.2 (iii), and Na v 1.6 (iv) subunits, but not Na x (i) subunits, expressed in rat brain tissues. AnkG: ankyrin-G; IgG-HC: immunoglobulin heavy chain; In: lysate input; IP: immunoprecipitation; KIF5B: kinesin family member 5B; mIgG1: mouse immunoglobulin isotype 1; mIgG2b: mouse immunoglobulin isotype 2b; MRLC: myosin regulatory light chain; myh: myosin heavy chain; Na v : voltage-sensitive sodium channel; Na x : nonvoltage-dependent sodium channel; PC: precleared.

Article Snippet: Polyclonal antibodies against Na v 1.2 (1:200; Alomone), Na v 1.3 (1:200; Alomone), Na v 1.6 (PN4; 1:200; Sigma), Na v 1.7 (1:200; Alomone), Na v 1.8 (1:200; Alomone), Na v 1.9 (1:200; Alomone), Na x (1:1000; Abcam), β-II spectrin (1:2000; Abcam), β-III spectrin (1 µg/mL; Santa Cruz Biotechnology), and GFP (1:1000; Invitrogen) were also used for immunoblotting.

Techniques: Western Blot, Immunoprecipitation