Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca 2+ channels

doi: 10.1073/pnas.1702991114

Figure Lengend Snippet: Antibodies used in the current study with corresponding dilutions and origin

Article Snippet: A list of antibodies used in the current study with corresponding dilutions and origin are presented in . table ft1 table-wrap mode="anchored" t5 Table S1. caption a7 Antigen Antibody Dilution Active zone proteins RBP-1 (RIM-binding protein-1) 316003 (SySy) 1:1,000 RBP-2 (RIM-binding protein-2) 316103 (SySy) 1:1,000 RBP-2 (RIM-binding protein-2) 4193 (custom) 1:1,000 RIM 1 (RIM1 central domains) R809 (custom) 1:2,000 RIM 1/2 (1α/2α N terminus + rabphillin) U1565 (custom) 1:2,000 Liprin α3 4396 (custom) 1:5,000 ELKS 1/2aB 4790 (custom) 1:500 CASK 75–000 (Neuromab) 1:1,000 Mint1 P932 (custom) 1;1000 Veli1,2,3 T813 (custom) 1:1,000 Munc13-1 126103 (SySy) 1:1,000 Ribeye Maxeiner et al. ( 32 ) 1:1,000 Ca 2+ channels Ca 2+ v1.2-α1C voltage-gated channel ACC-003 (Alomone) 1:200 Ca 2+ v1.3-α1D voltage-gated channel ACC-005 (Alomone) 1:200 Ca 2+ v1.4-α1D voltage-gated channel Gift from Frank Schmitz 1:1,000 Ca 2+ v-α2δ1 voltage-gated channel ACC-015 (Alomone) 1:500 Ca 2+ v-α2δ2 voltage-gated channel ACC-102 (Alomone) 1:500 Ca 2+ v-α2δ3 voltage-gated channel ACC-103 (Alomone) 1:500 Ca 2+ v-α2δ4 voltage-gated channel ACC-104 (Alomone) 1:500 Ca 2+ β1 voltage-gated channel ACC-106 (Alomone) 1:250 Ca 2+ β2 voltage-gated channel ACC-105 (Alomone) 1:250 Ca 2+ β3 voltage-gated channel ACC-108 (Alomone) 1:250 Ca 2+ β4 voltage-gated channel 75–054 (Neuromab) 1:250 Exocytosis proteins Syntaxin 1 HPC-1 (custom) 1:500 SNAP 25 71.1 (custom) 1:2,000 Synaptobrevin 2 69.1 (custom) 1:2,500 Synaptotagmin 1 41.1 (custom) 1:1,000 Loading control TUJ1 T2200 (Sigma) 1:5,000 Open in a separate window Antibodies used in the current study with corresponding dilutions and origin

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphoproteomics Study Based on In Vivo Inhibition Reveals Sites of Calmodulin‐Dependent Protein Kinase II Regulation in the Heart

doi: 10.1161/JAHA.113.000318

Figure Lengend Snippet: Benchmarking the CaMKII inhibitory mouse model. A, Western blots of cardiac phospholamban (PLN) Thr17 phosphorylation, CaMKIIδ autophosphorylation, and general PLN protein levels in set of 3 AC3‐I and 3 AC3‐C mice after isoproterenol stimulus (left). Western blots of Cacnb2 phosphorylation levels (Thr549, also referred to as Thr498 ) in another set of 2 mice. B, Quantitative MS profile of PLN peptides 15 to 25 confirms PLN phosphorylation is blunted in AC3‐I mice when compared with AC3‐C mice. The internal standard, a 1:1 mixture of the 2, shows the expected intermediate level. C, Icelogo of all observed motifs surrounding the phosphorylation site (7 amino acids up‐ and downstream, only peptides with a defined localized phosphosite were used [see Methods and Results for details]). Amino acid codes above the x axis show overrepresentation, whereas below indicates underrepresentation at a particular position. D, Icelogo of all motifs containing the minimal CaMKII consensus sequence [R/K]XX[pS/pT][notP]. E, Icelogo of all AC3‐I downregulated sites. All Icelogos generated with a P <0.01. CaMKII indicates calcium‐ and calmodulin‐dependent protein kinase II; Cacnb2, calcium channel beta 2.

Article Snippet: The following antibodies were used: anti‐phospho‐Cacnb2 antibodies made by Yenzyme, anti‐Cacnb2 (Thr549; Neuromab), anti‐phosphorylated‐CaMKII (Thr 287; Upstate), anti‐phospholamban (Upstate), and anti‐phospho‐PLN (Thr17; Santacruz).

Techniques: Western Blot, Sequencing, Generated

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Phosphoproteomics Study Based on In Vivo Inhibition Reveals Sites of Calmodulin‐Dependent Protein Kinase II Regulation in the Heart

doi: 10.1161/JAHA.113.000318

Figure Lengend Snippet: CaMKII affects the cardiac sarcomere at several distinct sites. A, Model of CaMKII‐affected sites in the cardiac myofibrils (z‐disc and a‐band) displaying described links with the sarcolemma, the costamere and the sarcoplasmic reticulum (SR). All proteins depicted, except calcineurin (CaN), were observed with a phosphorylation site in this study. The color codes are the same as in ; double‐colored proteins were observed with 2 oppositely regulated sites. The 3 boxed proteins (Bag3, Murc, and Myo18b) are established z‐disc proteins; however, their interaction partners have currently not been described. LTCC is L‐type calcium channel (Cacnb observed in our study); for other acronyms see Table S2. B, Immunolabeling of Csrp3 (red) revealed colocalization with the GFP‐tagged transgenic AC3‐I (green) at the sarcomere in left ventricle. Scale bar=10 μm. CaMKII indicates calcium‐ and calmodulin‐dependent protein kinase II; Obscn, obscurin; Plec, plectin; Tln, talin‐1; Sorbs1, sorbin and SH3 domain‐containing protein 1; Flnc, filamin‐C; Ahnak, AHNAK nucleoprotein isoform 1; Jph, junctophilin‐1; PLN, phospholamban; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; Cacnb2, calcium channel beta 2; Capzb, isoform 1 of F‐actin‐capping protein subunit beta; Igfn1, immunoglobulin‐like and fibronectin type III domain‐containing protein 1; Nexn, nexilin; Ablim1, isoform 4 of actin‐binding LIM protein 1; Myoz2, myozenin‐2; Ldb3, isoform 4 of LIM domain‐binding protein 3; Csrp3, cysteine‐ and glycine‐rich protein 3; Tcap, telethonin; Cobll1, Cordon‐bleu protein‐like 1; Lmod2, leiomodin‐2; Tpm, tropomyosin; Mybpc3, myosin‐binding protein C; Myl, myosin light chain; Myh, myosin heavy chain; Palld, palladin; Murc, muscle‐related coiled‐coil protein.

Article Snippet: The following antibodies were used: anti‐phospho‐Cacnb2 antibodies made by Yenzyme, anti‐Cacnb2 (Thr549; Neuromab), anti‐phosphorylated‐CaMKII (Thr 287; Upstate), anti‐phospholamban (Upstate), and anti‐phospho‐PLN (Thr17; Santacruz).

Techniques: Immunolabeling, Transgenic Assay, Binding Assay