Journal:

Article Title: Molecular basis of transient outward K + current diversity in mouse ventricular myocytes

doi: 10.1111/j.1469-7793.1999.00587.x

Figure Lengend Snippet: Ventricular membrane proteins (A: 180 μg loaded in each lane; B and C: 50 μg loaded in each lane) were fractionated on SDS-PAGE gels, transferred to PVDF membranes and immunoblotted with the polyclonal anti-Kv1.4 (A), anti-Kv1.2 (B) or anti-Kv2.1 (C) antibodies (see Methods). WT, TG and KO indicate ventricular membrane proteins prepared from adult wild-type, Kv4.2W362F-expressing and Kv1.4−/− mice, respectively. The arrows show the specific bands recognized by the antibodies.

Article Snippet: PVDF membrane strips were first incubated in 0.2% I-Block (Tropix) in PBS containing 0.1% Tween 20 (blocking buffer) for 1 h at room temperature, followed by overnight incubation at 4°C with polyclonal antibodies directed against Kv1.4 (Alamone Labs), Kv1.2 (Alamone Labs) and Kv2.1 (Upstate Biotechnology).

Techniques: SDS Page, Expressing

Journal: bioRxiv

Article Title: Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

doi: 10.1101/2022.09.12.507626

Figure Lengend Snippet: (a) Cartoon showing Q1/E1-YFP complex co-expressed with or without free PKA Cα subunit. (b) Representative immunoblots of lysates from HEK293 cells co-expressing Q1/E1-YFP with either empty pcDNA3.1 vector or free Cα. Anti-pKCNQ1 (top) detects phosphorylated KCNQ1-S27, anti-KCNQ1 (middle) detects total KCNQ1, and anti-Actin (bottom) detects total actin. (c) I Ks activation curves in CHO cells co-expressing Q1, E1-YFP with either empty pcDNA3.1 vector (black symbols, n = 13) or free PKA Cα (red symbols, n = 13). (d) Tail-decay times for currents recorded from cells co-expressing Q1/E-YFP + yotiao and either nano or free PKA Cα, or cells co-expressing Q1[S27A]/E1-YFP + yotiao and free PKA Cα. (e-h) Cartoon, immunoblots, I Ks activation curves, and population current densities of Q1/E1-YFP complex expressed with either nano (n = 10) or nanoCα ( n = 10). (i) Cartoon showing targeting of nanoCα to Q1/E1 complex via YFP tag on Q1 C-terminus. (j) Exemplar current traces I Ks traces from CHO cells co-expressing Q1-YFP/E1 with either nanoCα ( left ) or catalytically inactive nanoCα [T198A] mutant ( right ). (k) Population current densities (nano, n = 26; nanoCα, n = 19; nanoCα[T198A], n = 10).

Article Snippet: Membranes were pre-treated with 0.5% glutaraldehyde and re-blotted with rabbit anti-KCNQ1 antibody (1:1,000, Alomone labs, Israel) and rabbit anti-actin antibody (1:2000, Abcam, USA).

Techniques: Western Blot, Expressing, Plasmid Preparation, Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

doi: 10.1101/2022.09.12.507626

Figure Lengend Snippet: (a) Cartoon of FKBP/FRB heterodimerization strategy utilized for rapamycin-induced recruitment of engineered Cα to BBS-Q1-YFP/E1. (B) Exemplar flow cytometry contour plots showing surface expression (BTX-647 fluorescence) and CFP fluorescence in cells expressing BBS-Q1-YFP/E1 with FRB-Cα and FKBP-nano at times t = 0 ( left ), t = 6h ( middle ) and t = 24h ( right ) after rapamycin addition. (c) Normalized mean Q1 surface density (BTX-647 fluorescence) plotted as a function of time after rapamycin induction. (d) Normalized mean Q1 total expression (YFP fluorescence) plotted as a function of time after rapamycin induction. (e) Exemplar I Ks traces recorded in CHO cells co-expressing KCNQ1-YFP/KCNE1/Nano-FKBP-FRB-Cα incubated 20 hours either without ( left ) or with ( right ) rapamycin. (f) Mean current densities in CHO cells co-expressing KCNQ1-YFP/KCNE1/Nano-FKBP-FRB-Cα without rapamycin (black, n = 10) or after 20 h rapamycin incubation (red, n = 14). *** P < 0.001, paired t test. (g) Mean current densities in control cells co-expressing KCNQ1-YFP/KCNE1 without rapamycin (black, n = 8) or after 20 h rapamycin incubation (red, n = 9).

Article Snippet: Membranes were pre-treated with 0.5% glutaraldehyde and re-blotted with rabbit anti-KCNQ1 antibody (1:1,000, Alomone labs, Israel) and rabbit anti-actin antibody (1:2000, Abcam, USA).

Techniques: Flow Cytometry, Expressing, Fluorescence, Incubation, Control

Journal: bioRxiv

Article Title: Divergent regulation of KCNQ1/E1 by targeted recruitment of protein kinase A to distinct sites on the channel complex

doi: 10.1101/2022.09.12.507626

Figure Lengend Snippet: (a) Top , schematic of Q1 showing positions of Ser and Thr residues where phosphorylation was increased when nanoCα was targeted to Q1 C-terminus. Bottom , relative abundance of phosphorylated KCNQ1-YFP peptides identified using mass spectrometry in cells co-expressing nano (black), nanoCα (red), or free Cα (cyan). (b) Exemplar CDF plots showing channel surface density in cells expressing WT BBS-Q1-YFP ( left ), BBS-3S/A-YFP ( middle ), or BBS-3S/D-YFP ( right ) in the absence (black traces) or presence (red traces) of nanoCα. (c) Channel surface density (BTX-647 fluorescence) in cells expressing WT BBS-Q1-YFP, BBS-3S/A-YFP, or BBS-3S/D-YFP in the presence of either nano or nanoCα. WT BBS-Q1-YFP (nano, n = 15,411, N = 2; nanoCα, n = 9,053, N = 2). BBS-3S/A-YFP (nano, n = 6,158, N = 2; nanoCα, n = 4,798, N = 2). BBS-3S/D-YFP (nano, n = 11,823, N = 2; nanoCα, n = 5,685, N = 2).

Article Snippet: Membranes were pre-treated with 0.5% glutaraldehyde and re-blotted with rabbit anti-KCNQ1 antibody (1:1,000, Alomone labs, Israel) and rabbit anti-actin antibody (1:2000, Abcam, USA).

Techniques: Phospho-proteomics, Mass Spectrometry, Expressing, Fluorescence