MA3-920

antibody from Invitrogen Antibodies

Targeting: CACNA1S CACNL1A3, Cav1.1, HOKPP, hypoPP, MHS5

Provider product page for MA3-920

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [79]
Comments [0]
Validations
Immunohistochemistry [1]
Flow cytometry [1]
Other assay [26]

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Product number: MA3-920 - Provider product page
Provider: Invitrogen Antibodies
Product name: CaV1.1 Monoclonal Antibody (1A)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-920 detects 1,4-dihydropyridine (DHP) receptor alpha-1 subunit in human, rat, mouse, guinea pig and rabbit skeletal muscle. The DHP Receptor alpha-1 protein is also known as CACNA1S or Cav1.1 alpha-1 subunit.
Antibody clone number: 1A
Concentration: 1 mg/mL

CACNA1S protein structure - MA3-920 shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 Fat1 expression at late stages of muscle differentiation. ( A ) Fat1 expression was visualized in E13.5 embryos or in neonate (P0) muscle by beta-galactosidase staining or by in situ hybridization with a Fat1 3'UTR RNA probe. ( B - D ) Immunolocalization of FAT1 (anti-FAT1-ICD from [35] , green) was performed in E12.5 mouse embryo ( C ), and on adult ( B, D ) muscle fibers on longitudinal muscle cryosections from wild type ( B , C 1-3 , D 1,4 ), from Fat1 DeltaTM/DeltaTM embryos ( C 4-6 ), and from Fat1 LacZ/LacZ ( D 2-3 , D 5-6 ) mice, combined with either antibodies against alpha-actinin (red, B 5 ), DHPR (Cacna1s) (red, B 2,3 ), or RyR (red, B 4 ), or with Phalloidin (red, C, D ). In D , Green channel images (FAT1) were first captured with either identical exposure time between wild type and mutants ( D 1,4 and D 2,5 , 421 ms), or with longer exposure time ( D 3,6 , 2222 ms). This indicates that the epitope detected by the anti-FAT1-ICD antibody (from ref [35] ) is present in reduced but detectable amounts in Fat1 LacZ/LacZ muscles. This observation was made when Fat1 LacZ/LacZ mice (n = 2 at P0; and n = 3 at adult stages) displayed severe muscle defects at the stage of dissection, indicating that levels of FAT1 protein inversely correlate with phenotype severity. Scale bars: ( B - D ) 4 um, ( C ) 6 um.

Supportive validation

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Experimental details: Fig 2 Misexpression of triad proteins and expansion of the T-tubule compartment in Ehd1- heterozygous muscle. (A) BIN1 and DHPR levels are increased +2.25 and +5.4 fold in Ehd1 +/- quadriceps muscle compared to WT correlating with the increase in T-tubule structures. Gel code bands are shown as a loading control (LC). (B) Junctophilin 2 (JP2) protein levels were decreased 13-fold in Ehd1+/- quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (C) Junctophilin 1 (JP1) protein levels were similar in Ehd1+/- quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (D) Ultrastructural analysis reveals ectopic (dotted arrow) and elongated (arrow) T-tubules in 8-week-old Ehd1- heterozygous muscle ( Ehd1+/- ) stained with potassium ferricyanide to color the T-tubule structures black. (E) Ehd1- heterozygous muscle contains duplicated triads containing 2 T-tubules (black arrows) and 3 sarcoplasmic reticulum (SR) in 1 triad unit. Scale 0.5mum. (F) Ehd1- heterozygous muscle stained with potassium ferricyanide, outlines duplicated T-tubule structures (two black arrows). Scale 0.5mum. (G) Ultrastructural analysis of 2-D images reveals increased tubule abnormalities in Ehd1- heterozygous muscle, 12.5%, compared to 1.7% in control muscle (n>400 structures per genotype, p = 0.04).

Supportive validation

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Experimental details: Figure 6. Thbs4 enhances stabilizing proteins at the sarcolemma. ( A ) Immunofluoresence (green) detection of delta-, alpha-, beta-, and gamma-sarcoclycan (SGC), beta-dystroglycan (beta-DG), utrophin (Utro.), dystrophin (Dystro.) and beta1D-integrin in littermates of three month-old WT, Thbs4-Tg, Sgcd -/- and Sgcd -/- Thbs4-Tg quadriceps. Representative images of 4 mice per genotype are shown. Scale bar = 25 mum. ( B ) Representative Western blots of sarcolemmal protein extracts (upper) or total cytoplasmic protein extracts (lower) from the quadriceps of the indicated groups of mice for the indicated proteins (n = 4-5 biological replicates). Abbreviations: Utro, utrophin; Dystro, dystrophin; Dysfer, dysferlin; alpha-DG, alpha-dystroglycan; beta-DG, beta-dystroglycan; delta-SCG, delta-sarcoglycan; alpha-SCG, alpha-sarcoglycan; beta-SGC, beta-sarcoglycan; beta1D-, alpha7- and alpha5- integrin. The red boxes show increased protein levels. Also see Figure 6--figure supplement 1 for replicates. ( C ) Representative immunoblotting for structural components of the DGC- and integrin-associated protein complexes in sarcolemmal preparations from Thbs4 -/- and WT quadriceps at four months of age (n = 4 biological replicates). The burgundy-boxed areas show reduced protein levels. Ponceau staining of a nonspecific band and dihydropyridine receptor alpha1 (Cav1.1) were used as loading controls for sarcolemmal protein extracts; Gapdh was used as loading control for total cell protein extrac

Supportive validation

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Experimental details: Fig 1 Disordered T-tubules in Ehd1- heterozygous muscle. Myofibers were immunostained with anti-BIN1 (red) and anti-DHPR (green) antibodies. Representative myofibers are shown. Ehd1- heterozygous ( Ehd1+/- ) fibers displayed disorganized (white arrowhead) and aggregated (white arrow) T-tubule structures in 27% of myofibers, marked by DHPR, also evidenced in DIC images compared to 0% in control fibers. Ehd1 -heterozygous muscle with extensive BIN1 fluorescence extending beyond DHPR staining (yellow arrowhead). Scale 5mum.

Supportive validation

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Experimental details: Figure 7. Skeletal muscle specific ATF6alpha overexpression drives ER stress and intracellular vesicular expansion, but not protection against MD. ( A ) Schematic diagram of the transgene (Tg) used to overexpress ATF6alpha in skeletal muscle. ( B ) Western blot analysis for Thbs4, ATF6alpha, BiP, PDI and calreticulin (Calret.) expression in quadriceps (Quad) from WT, Thbs4 Tg and ATF6alpha Tg mice at six weeks of age. Gapdh is a processing and loading control (n = 6 biological replicates). ( C ) Masson's trichrome-stained histological sections from Quad of WT and ATF6alpha-Tg littermates at six weeks of age. Representative images of 5 mice per genotype are shown. Scale bar = 100 mum. ( D ) Transmission electron micrographs in Quad from WT and ATF6alpha-Tg mice at six weeks of age. The white arrows show dramatic expansion of ER and associated vesicles throughout the cell and especially in the sub-sarcolemmal region. Representative images of 2 mice per genotype are shown. Scale bar = 2 mum. ( E ) Masson's trichrome stained histological sections of Quad from Sgcd -/- and Sgcd -/- ATF6alpha Tg mice at six weeks of age. Representative images of 5 mice per genotype are shown. Scale bar = 100 mum. ( F ) Quantitation of serum CK levels (units/liter) in the indicated genotypes of mice shown in the legend below the graph at six weeks of age. n = 10 mice for WT and n = 8 mice for the remaining genotypes. *p

Supportive validation

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Experimental details: Figure 2 ARG ingestion inhibits ECC -induced proteolysis of Ca 2+ regulatory proteins. For the protocols of ARG ingestion and ECC , see legend of Figure 1 . (A and E) immunoblot analyses of RyR, DHPR , JP 1, and CSQ 1 in tibialis anterior muscles. RyR, DHPR , and JP 1 were analyzed on the same membrane. For RyR and DHPR , in addition to full-length proteins, faster migrating bands were observed and most likely corresponded to degraded proteins. Total proteins on the membrane, which were used as a loading control, were visualized with Coomassie blue R staining. Positions of molecular mass makers are indicated on the left. (B, C, D, and F) means +- SEM ( n = 7 for each muscle) of the contents of full-length RyR (arrow), DHPR , JP 1, and CSQ 1, respectively. The contents of four proteins investigated were evaluated relative to total proteins. The results are expressed as percentages of the values observed in rested muscles from CON rats. * P < 0.05, versus rested muscles within rats; + P < 0.05, versus ECC muscles from CON rats. ARG , l -arginine; CON , control; CSQ 1, calsequestrin-1; DHPR , dihydropyridine receptor; ECC , eccentric contraction; JP 1, junctophilin-1; RyR, ryanodine receptor.

Supportive validation

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Experimental details: Figure 8 In vitro S-nitrosylation of calpain-1 inhibits Ca2+-induced proteolysis of Ca2+ regulatory proteins. (A) immunodetection of GSNO-induced S-nitrosylation of calpain-1 in tibialis anterior muscles. To check the assay specificity and ascertain whether GSNO treatment can result in S-nitrosylation of calpain-1, SNO-RAC was performed in the presence (+) and absence (−) of GSNO and Cu-Asc treatment. Positions of molecular mass markers are indicated on the left. (B) immunoblots of RyR, DHPR, and JP1. Muscle homogenates were incubated in the presence (+) and absence (−) of GSNO and Ca2+. Total proteins on the membrane, which were used as a loading control, were visualized with Coomassie blue R staining. (C, D and E) means ± SEM (n = 5 for each treatment) of the contents of full-length RyR (arrow), DHPR, and JP1, respectively. The contents of the three proteins were evaluated relative to the total proteins. The results are expressed as percentages of the values in samples treated without GSNO and Ca2+. a P < 0.05, versus T1. b P < 0.05, versus T2. c P < 0.05, versus T3. GSNO, S-nitrosoglutathione; Cu-Asc, CuCl and sodium ascorbate; DHPR, dihydropyridine receptor; JP1, junctophilin-1; RyR, ryanodine receptor; SNO-RAC, resin-assisted capture of S-nitrosothiols; T, treatment.

Supportive validation

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Experimental details: Figure 3 Comparison of healthy and Duchenne muscular dystrophy (DMD) MyoT from hiPSCs and tissues at the protein level. (A) hiPSC-derived MyoT immunolabelling of alpha-actinin (red) and nuclei (DAPI, blue) in healthy (left) and DMD cells (right). (B) Representative western blots and related quantifications of DMD, SGCA, SGCG, myosin heavy chains, CACNA1S, and RYR1 from protein extracts in healthy and DMD hiPSC-derived and tissue-derived MyoT (X: 0.25 mug of total protein was used in hiPSC-derived MyoT instead of 7 mug in tissue-derived MyoT--* P value

Supportive validation

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Experimental details: Figure 9 FAT1 protein and RNA levels are mis-regulated in human foetal FSHD tissues. ( A ) Immunolocalization of FAT1 (Rb-1465 anti FAT1-ICD, green) and DHPR (Cacna1s, magenta) in longitudinal sections from human quadriceps biopsies from a control (top) or and FSHD (F1, bottom) foetus with 1.5 D4Z4 repeats. ( B ) qPCR analysis of FAT1 mRNA levels in quadriceps (3 left graphs) and deltoid muscles (middle graph) and in brain (right graph), comparing respectively with age-matched control foetuses (blue bars), a 26 weeks old FSHD1 foetus (F1) harbouring 1.5 D4Z4 repeats in the 4q35 region (dark red bars), a 16 weeks old FSHD1 foetus harbouring 4.3 D4Z4 repeats at 4q35 region (F2), and twin FSHD1 foetuses aged 28 weeks, with 7 D4Z4 repeats. ( C ) Analysis of the regulatory status of the promoter region by Chromatin immunoprecipitation. The respective level of the following histone marks: H3K27me3 (silenced chromatin; C-left ), and H3K4m3 (promoter active; C-right ), in muscle extracts from four age matched controls (ct1 to 4) or four FSHD1 foetuses (F1 to F4) are shown. Relative quantities were normalized with the level of histone marks at the promoter of the GUSB gene as internal control, and expressed as % of control 1 (ct1). Scale bars: ( A ) 50 um.

Supportive validation

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Experimental details: Fig. 5 ANO1 is localised in the zebrafish skeletal muscle surface membrane. Representative images of normal zebrafish myotubes double immunolabelled for DHPRalpha 1S subunit (anti-alpha 1S ) and ANO1 (anti-ANO1) or ANO5 (anti-ANO5) isoforms showing their respective subcellular distribution. a ANO1 is expressed in the sarcolemma (centre image) and does not co-localise with DHPRalpha 1S (left image) in the triads (merged image). Scale bar, 5 um. Fluorescence intensity profile (bottom) obtained by measuring the average fluorescence intensity along a horizontal line on the merged image demonstrates regular t-tubular periodicity of the DHPRalpha 1S (red) but a complete lack of a structured ANO1 signal (green). b Double immunofluorescence labelling of DHPRalpha 1S (left image) and ANO5 (centre image) under similar conditions clearly revealed anti-ANO5 signal on both sides of the t-tubular membrane (merged image) and thus indicating SR membrane localisation of ANO5. Fluorescence intensity profile displays distinct but regular spatial periodicity with one red peak (DHPR signal) flanked by two green peaks (ANO5 signal)