mouse anti syx1  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Regulation of presynaptic Ca 2+ channel abundance at active zones through a balance of delivery and turnover

doi: 10.1101/2022.03.14.484315

Figure Lengend Snippet: (A) Representative images of control NMJs with both Ib and Is motoneurons co-innervating muscle 1 (top left), or muscle 1 with only Ib innervation (top right), and Is and cac -flipped Ib neurons co-innervating muscle 1 (bottom left) or cac -flipped Ib neurons with no Is present (bottom right). The flip-reporter tdTomato (magenta) and HRP (neuronal membranes; purple) were imaged prior to fixation (left panels). After fixation, NMJs were immunostained for BRP (red) and GluRIII (green) to label AZs and PSDs, respectively (right panels). (B, C) Average traces and evoked amplitudes (peak current, nA) from the indicated genotypes (co-innervated controls: orange, 42.11±1.757, n=8 NMJs from 6 animals; co-innervated Cac Ib-flp : green, 15.13±1.607, n=15 NMJs from 12 animals, p<0.0001; Ib-only controls: blue, 32.32±2.329, n=6 NMJs from 6 animals; Ib- only Cac Ib-flp : gold, 5.749±0.9544, n=5 NMJs from 5 animals, p<0.0001). (D) Neuronal (HRP- stained) area of Ib synapses (co-innervated controls: orange, 219.1±15.15, n=8 NMJs from 6 animals; co-innervated Cac Ib-flp : green, 190.4±21.12, 9 NMJs from 8 animals; Ib-only controls: blue, 207.8±22.4, n=6 NMJs from 6 animals; Ib-only Cac Ib-flp : gold, 189±24.59, n=5 NMJs from 5 animals). (E) Number of AZs formed by Ib synapses (co-innervated controls: orange, 224.3±16.88; co-innervated Cac Ib-flp : green, 255.1±10.13; Ib-only controls: blue, 245.8±14.73; Ib- only Cac Ib-flp : gold, 220.4±13.47). (F) Some NMJs lacking Cac display ectopic filopodia extending from boutons (white arrowheads). (G) Representative images of cac -flipped NMJs stained with anti-BRP (magenta) and anti-RBP (green). (H) Quantification of BRP abundance. Each point represents the average fluorescence intensity across the AZ population of a single NMJ (CTRL: orange, 27736±900.2, n=12 NMJs from 6 animals; Cac Ib-flp : green, 25943±843.7, n=12 NMJs from 6 animals). (I) Quantification of RBP abundance (CTRL: orange, 19910±439.2; Cac Ib-flp : green, 23593±530.6, p<0.0001). (J) Representative images of cac -flipped animals stained for SYX1 (magenta) and CPX (green). (K) Quantification of SYX1 abundance. Each point represents the average fluorescence intensity across the bouton population of a single NMJ (CTRL: orange, 16028±683.5, n=9 NMJs from 5 animals; Cac Ib-flp : green, 17365±643.4, n=12 NMJs from 6 animals). (L) Quantification of CPX abundance (CTRL: orange, 18810±509.4; Cac Ib-flp : green, 17961±663.5).

Article Snippet: Primary antibodies used in this study were mouse anti-BRP at 1:500 (Nc82 DSHB, Iowa City, IA), rabbit anti-RBP at 1:500 (provided by Stephan Sigrist), mouse anti-SYX1 at 1:100 (8C3; DSHB), rabbit anti GluRIII at 1:500 , and anti-CPX at 1:500 ( ).

Techniques: Staining, Fluorescence

Journal: bioRxiv

Article Title: Regulation of presynaptic Ca 2+ channel abundance at active zones through a balance of delivery and turnover

doi: 10.1101/2022.03.14.484315

Figure Lengend Snippet: (A) Representative images of Cac-GFP (green) and BRP (magenta) at muscle 4 for endogenously C- terminally tagged Cac-GFP controls ( cac GFP(C) ,C155/cac GFP(C) ) and Cac overexpression animals ( cac GFP(C) ,C155/cac GFP(C) ,UAS-Cac-GFP ). (B) Quantification of AZ Cac intensity in control versus Cac overexpression NMJs. Each point represents the maximum Cac-GFP pixel intensity of a single AZ (Control: 14617±130.8, n=3663 AZs from 10 NMJs from 6 animals; Cac OE: 14383±148.2, n=3145 AZs from 10 NMJs from 6 animals). (C) Distribution of AZ Cac-GFP abundance across the AZ population in control versus Cac overexpression NMJs. (D) Representative images of endogenously tagged Cac-GFP (green) and overexpressed Cac- tdTomato (magenta) at AZs in control ( cac GFP ,C155 ) versus Cac-tdTomato overexpression ( cac GFP ,C155;UAS-Cac-tdT ). (E) Quantification of AZ Cac-GFP intensity with and without Cac- tdTomato overexpression (Control: 15339±232, n=1613 AZs, Cac-tdT: 4477±123.9, n=850AZs, p<0.0001). (F, G) Average traces and evoked peak currents (nA) at muscle 6 in Cac overexpression and control animals (Control: 24.45±2.433, n=9 NMJs from 5 animals; Cac OE: 30.11±3.046, n=9 NMJs from 5 animals). (H, I) Quantifications and representative images of western blots of adult head extracts from cac GFP/Df heterozygotes and cac GFP controls (n=11 samples, extracts from 5 adult heads per sample). Each point in H represents Cac intensity in one lane ( cac GFP : 25092±2146; cac GFP/Df : 13055±1403, p<0.001). Each point in I represents SYX1 intensity in one lane ( cac GFP : 224900±11239; cac GFP/Df : 214889±8696). Representative images for Cac and SYX are shown above each quantification. (J) Representative images of endogenous Cac- GFP (green) and BRP (magenta) at muscle 4 AZs in cac GFP/Df heterozygotes and cac GFP controls. Ib and Is terminals are labeled in white. (K) Quantification of average Cac-GFP AZ abundance per MN4-Ib NMJs ( cac GFP : 15105±486.3, n=19 NMJs from 7 animals; cac GFP/Df : 13617±365.9, n=21 NMJs from 7 animals, p<0.05). (L) Histogram of Cac-GFP intensity across the AZ population in cac GFP/Df heterozygotes and cac GFP controls at MN4-Ib NMJs. (M) BRP-positive AZ number per MN4-Ib NMJ ( cac GFP : 243.4±17.05; cac GFP/Df : 260.3±9.963). (N) Quantification of average AZ Cac-GFP intensity per Is innervation of muscle 4 NMJs ( cac GFP : 14621±747.5, n=11 NMJs from 7 animals; cac GFP/Df : 10219±601 n=12 NMJs from 7 animals, p<0.001). (O, P) Average traces and evoked peak currents (nA) at muscle 6 in cac GFP/Df heterozygotes and cac GFP controls ( cac GFP : 41.96±3.879, n=9 NMJs from 5 animals; cac GFP/Df : 16.7±1.984, n=8 NMJs from 7 animals, p<0.0001).

Article Snippet: Primary antibodies used in this study were mouse anti-BRP at 1:500 (Nc82 DSHB, Iowa City, IA), rabbit anti-RBP at 1:500 (provided by Stephan Sigrist), mouse anti-SYX1 at 1:100 (8C3; DSHB), rabbit anti GluRIII at 1:500 , and anti-CPX at 1:500 ( ).

Techniques: Over Expression, Western Blot, Labeling

Journal: bioRxiv

Article Title: Regulation of presynaptic Ca 2+ channel abundance at active zones through a balance of delivery and turnover

doi: 10.1101/2022.03.14.484315

Figure Lengend Snippet: (A) Representative images of BRP and endogenously tagged Cac-GFP at AZs in controls, brp Df/+ heterozygotes and pan- neuronally ( elav - GAL4 ) expressed BRP RNAi. (B) Quantification of single AZ BRP intensity, with average AZ BRP intensity listed as a percent of control above each genotype (control: 20161±210.7, n=1779 AZs; brp Df/+ : 13534±176.7, n=1350 AZs, p<0.0001; BRP RNAi: 4806±79.91, n=1140 AZs, p<0.0001). (C) Representative image of Western blots of adult head extracts from control, brp Df/+ heterozygotes, and pan-neuronally expressed BRP RNAi animals stained for SYX1 (loading control), Tubulin, and BRP. (D) Quantification of BRP intensity in Western blots of the indicated genotypes. Each point represents BRP intensity in one lane, with BRP intensity normalized to the SYX1 loading control. Percent of protein abundance compared to control (100%) is shown above each genotype (control: 1.0±0.02912, n=9 lanes; brp Df/+ : 0.6518±0.03862, n=10 lanes, p<0.0001; BRP RNAi: 0.1366±0.02117, n=5 lanes, p<0.0001). (E) Quantification of endogenously tagged Cac-GFP intensity at single AZs in controls, brp Df/+ heterozygotes, and pan-neuronally expressed BRP RNAi (control: 23268±232.4, n=1757 AZs; brp Df/+ : 22788±217, n=1403 AZs; BRP RNAi: 17959±259, n=1140 AZs, p<0.0001). (F, G) Average traces and quantified evoked peak currents (nA) in control and brp Df/+ heterozygotes at muscle 6 (control: 26.78±1.632, n=11 NMJs; brp Df/+ : 28.71±3.105, n=13 NMJs).

Article Snippet: Primary antibodies used in this study were mouse anti-BRP at 1:500 (Nc82 DSHB, Iowa City, IA), rabbit anti-RBP at 1:500 (provided by Stephan Sigrist), mouse anti-SYX1 at 1:100 (8C3; DSHB), rabbit anti GluRIII at 1:500 , and anti-CPX at 1:500 ( ).

Techniques: Western Blot, Staining