α 1c alomone  (Alomone Labs)

Journal: eLife

Article Title: A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary Ca V β subunits

doi: 10.7554/eLife.49253

Figure Lengend Snippet: ( a ) Cartoon of experimental strategy. BBS-α 1C was transfected in HEK293 cells with each YFP-Ca V β and either CFP or nb.F3-P2A-CFP. ( b ) Exemplar flow cytometry dot plot of cells transfected with BBS-α 1C , Ca V β 1 and CFP (left) or nb.F3 (right). ( c ) Cumulative distribution histogram of Alexa-647 (left) or YFP fluorescence (right) from CFP (black) or nb.F3 (red) expressing cells. YFP-positive cells (n > 5,000 cells/experiment) were selected for analysis; the threshold for 647 labeling and YFP fluorescence above background is represented with the dashed line. ( d ) Summary flow cytometry data of surface (647, filled) and total (YFP, patterned) levels of BBS-α 1C . Data from nb.F3 was normalized to CFP control group. N = 4 separate experiments, error bars, s.e.m. ( e ) population I-V curves from whole-cell patch clamp measurements in HEK293 cells expressing α 1C , Ca V β 2a , and CFP (black, n = 9) or nb.F3 (red, n = 12). Data are means ± s.e.m.

Article Snippet: Primary antibodies and working dilutions were as follows: α 1C : Alomone, 1:1000; UC Davis/NIH NeuroMab Facility, clone N263/31, 1:200.

Techniques: Transfection, Flow Cytometry, Fluorescence, Expressing, Labeling, Patch Clamp

Journal: eLife

Article Title: A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary Ca V β subunits

doi: 10.7554/eLife.49253

Figure Lengend Snippet: ( a ) Schematic of experimental design. HEK293 cells were transfected with BBS-α 1C , YFP-Ca V β, and either nb.F3, nb.F3-Nedd4L or nb.F3-Nedd4L[C942S]. ( b ) Exemplar flow cytometry dot plot of cells transfected with BBS-α 1C , YFP-Ca V β 1b and nb.F3 (left), nb.F3-Nedd4L (middle) or nb.F3-Nedd4L[C942S] (right). ( c,d ) Histogram of YFP ( c ) and Alexa-647 ( d ) fluorescence from samples in ( b ) (left) and summary data from N = 3 separate experiments (right). YFP-positive cells (n > 5,000 cells per experiment) were selected for analysis, the threshold for 647 labeling above background is represented with the dashed line. *p<0.05 compared with control, one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Primary antibodies and working dilutions were as follows: α 1C : Alomone, 1:1000; UC Davis/NIH NeuroMab Facility, clone N263/31, 1:200.

Techniques: Transfection, Flow Cytometry, Fluorescence, Labeling

Journal: eLife

Article Title: A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary Ca V β subunits

doi: 10.7554/eLife.49253

Figure Lengend Snippet: ( a ) Population I-V curves from HEK293 expressing α 1C + β 1b + α 2 δ−1 with either nb.F3 (black, I peak, 0mV = −48.4 ± 8.4 pA/pF, n = 12) or Ca V -aβlator (red, I peak, 0mV = −0.93 ± 0.16 pA/pF, n = 8). ( b-d ) Same format as ( a ) for cells expressing reconstituted Ca V 1.3 ( b ), Ca V 2.1 ( c ), or Ca V 2.3 ( d ) channels. Data are means ± s.e.m.†p<0.01 compared with control, unpaired, two-tailed Student’s t-test.

Article Snippet: Primary antibodies and working dilutions were as follows: α 1C : Alomone, 1:1000; UC Davis/NIH NeuroMab Facility, clone N263/31, 1:200.

Techniques: Expressing, Two Tailed Test

Journal: eLife

Article Title: A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary Ca V β subunits

doi: 10.7554/eLife.49253

Figure Lengend Snippet: ( a ) Confocal images (top) and exemplar traces from whole-cell recordings of uninfected guinea pig cardiomyocytes (left), or infected with adenovirus expressing either Ca V -βlator (middle) or nb.F3-Nedd4L[C942S] (right). Scale bar 0.2nA, 10 ms. ( b ) Population I-V curves from cardiomyocytes expressing Ca V -βlator (red), nb.F3-Nedd4L[C942S] (green), or an uninfected control (black). ( c ) Left, exemplar confocal images of cardiomyocytes fixed and immunostained with α 1C (green) and ryanodine receptor (RyR2, magenta) antibodies. Yellow box indicates region of high-zoom merge image.Right, co-localization between α 1C and RyR in uninfected cardiomyocytes (gray, PCC = 0.47 ± 0.02, n = 15), and those expressing either Ca V -aβlator (red, PCC = 0.24 ± 0.02 n = 19), or nb.F3-Nedd4L[C942S] (green, PCC = 0.50 ± 0.01, n = 17). ( d ) Left, exemplar confocal images of fixed cardiomyocytes immunostained with α 1C (green) and Rab7 (magenta) antibodies. Yellow box indicates region of high-zoom merge image. Right, colocalization between α 1C and Rab7 in uninfected cardiomyocytes (gray, PCC = 0.29 ± 0.02, n = 16), and those expressing either Ca V -aβlator (red, PCC = 0.42 ± 0.02, n = 18), or nb.F3-Nedd4L[C942S] (green, PCC = 0.30 ± 0.03, n = 16). ( e ) Pulldown of α 1C in HEK293 cells expressing α 1C , β 1b and either CFP, nb.F3, Ca V -aβlator, or nb.F3-Nedd4L-[C942S]. Top, probing pulldown with α 1C antibody. Bottom, same blot stripped and re-probed with ubiquitin antibody. ( f ) Quantification of four separate experiments, as performed in ( e ). Data are means ± s.e.m for each point. *p<0.05 compared to control, one-way ANOVA with Tukey’s multiple comparison test. ( g ) Pulldown of Ca V β 1b , as in ( e ). Left, probing with Ca V β 1b . Right, same blot stripped and re-probed with ubiquitin antibody. ( h ) Cartoon illustrating Ca V -aβlator-induced relocation of Ca V 1.2 from dyads to Rab7-positive late endosomes in cardiomyocytes.

Article Snippet: Primary antibodies and working dilutions were as follows: α 1C : Alomone, 1:1000; UC Davis/NIH NeuroMab Facility, clone N263/31, 1:200.

Techniques: Infection, Expressing

Journal: eLife

Article Title: A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary Ca V β subunits

doi: 10.7554/eLife.49253

Figure Lengend Snippet: ( a ) Comparison of total α 1C levels in uninfected cardiomyocytes (gray, n = 15) and those infected with nb.F3-Nedd4L (red, n = 19). ( b ). Comparison of total β 2 levels in uninfected cardiomyocytes (gray, n = 15) and those infected with nb.F3-Nedd4L (red, n = 16), normalized to uninfected controls. Data are means ± s.e.m. ( c ) Left, exemplar confocal images of uninfected (top) or F3-Nedd4L-infected (bottom) guinea pig cardiomyocytes, fixed and immunostained with antibodies towards α 1C (left) and LAMP1 (middle). Right, colocalization between α 1C and Rab5 in uninfected cardiomyocytes (gray, n = 7) and those expressing F3-Nedd4L (red, n = 7). Data are means ± s.e.m. ( d ) as in ( c ), showing immunostaining and colocalization analysis of cardiomyocytes immunostained against α 1C and Rab5.

Article Snippet: Primary antibodies and working dilutions were as follows: α 1C : Alomone, 1:1000; UC Davis/NIH NeuroMab Facility, clone N263/31, 1:200.

Techniques: Infection, Expressing, Immunostaining

Journal: eLife

Article Title: A potent voltage-gated calcium channel inhibitor engineered from a nanobody targeted to auxiliary Ca V β subunits

doi: 10.7554/eLife.49253

Figure Lengend Snippet:

Article Snippet: Primary antibodies and working dilutions were as follows: α 1C : Alomone, 1:1000; UC Davis/NIH NeuroMab Facility, clone N263/31, 1:200.

Techniques: Recombinant, Clone Assay, Mutagenesis, Plasmid Preparation, Software