Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: Distribution of store operated calcium channels in Cav +/+ and Cav∼ ∼ tissues. At left are micrographs of Cav +/+ and at right of Cav∼ ∼ tissues, cut with the LM in cross section. The top row of sections shows staining for Cav-1 and the second row shows staining for Orai1 in store operated channels. The third row shows the merged images. Note that Cav-1 Orai 2 stainings in Cav +/+ tissues are punctate at the cell membrane and are well co-localized. In Cav∼ ∼ tissues Cav-1 is absent and Orai 2 staining appears less clearly punctate. Length bars are 10 μM.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) Effects on contraction frequencies of LM segments from two successive exposures of control (Cav +/+ ) and Cav-1 knockout (Cav −/− ) to Ca 2+ Krebs solution with 0.1 mM EGTA. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. When there were significant differences between Cav +/+ and Cav −/− segments the bars for the Cav +/+ tissues are red. ( B ) Effect on contraction amplitudes in the experiments shown in Fig. 1A, with data from the final responses to 10 −5 M CCH. . Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. When there were significant differences between Cav +/+ and Cav −/− segments in spontaneous contraction amplitudes the bars for the Cav +/+ tissues are red but difference in amplitudes of contractions to CCH are in red for Cav −/− tissues. Responses to CCH after exposure to 0.1 mM EGTA are shown by perpendicular hatching and responses after exposure to 1 mM EGTA are shown with diagonal hatching.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) Effects on contraction frequencies of LM segments from two successive exposures of control (Cav +/+ ) and Cav-1 knockout (Cav −/− ) to Ca 2+ Krebs solution with 0.1 mM EGTA. In these experiments, either BayK 8644 or nicardipine (both at 10 −6 M) was added with the Ca 2+ -free medium. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. The significantly higher frequencies in Cav −/− tissues are indicated by red bars. ( B ) Effect on contraction amplitudes in the experiments shown in (A) , with data from the final responses to 10 −5 M CCH. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. When there were significant differences between Cav +/+ and Cav −/− segments in spontaneous contraction amplitudes after block of L-type Ca 2+ channels the bars are in red for Cav +/+ tissues.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out, Blocking Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) and ( B ) Effects on contraction frequencies ( A ) and amplitudes ( B ) of LM segments, during two successive exposures of control Effects on contraction frequencies ( A ) and amplitudes ( B ) of LM segments, during two successive exposures of control (Cav +/+ ) and Cav-1 knockout (Cav −/− ) to Ca 2+ Krebs solution with 0.1 mM EGTA. Some tissues had received an exposure to 10 μM CCH just prior to the first wash in Ca 2+ solution. There were no significant differences between Cav +/+ and Cav −/− tissues.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) and ( B ) Responses to 10 μM CCH of Cav +/+ and Cav −/− tissues exposed to CCH in normal Krebs solution ( A ) and in Ca 2+ -free solution ( B ). Phasic responses were measured at the maximum and tonic responses were measured 2 min. after the addition of CCH. In medium with normal Ca 2+ concentrations there were no significant differences in responses to CCH, but in Ca 2+ -free medium responses of tissues without caveolae were all significantly greater.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) Effects on contraction frequencies of LM segments, during three successive exposures of control (Cav +/+ ) (top panel) and Cav-1 knockout (Cav −/− ) (bottom panel) tissues to Ca 2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca 2+ -free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼ 6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca 2+ -free medium were higher in Cav +/+ tissues than in Cav −/− tissues, only those measured during the third wash with BayK 8644 and the second and third washes with nicardipine were significantly so, indicated by red bars. ( B ) Effects on contraction amplitudes of LM segments, during three successive exposures of control (Cav +/+ ) (top panel) and Cav-1 knockout (Cav −/− ) (bottom panel) tissues to Ca 2+ Krebs solution with 0.1 mM EGTA. Before the first exposure, 10 μM CCH was applied to all tissues for 2 min. and reapplied for 2 min. after each wash in Ca 2+ -free medium. CCH was washed out before the following exposure to this medium. In some tissues, either BayK 8644 or nicardipine (both 10∼ 6 M) were added with the calcium-free solution. Significant differences between the first and second exposures and initial values are indicated by #, the number symbol, and between comparable tissues by asterisks. Although nearly all mean values in Ca 2+ -free medium were higher in Cav +/+ tissues than in Cav −/− tissues, only those measured during the second and third washes with nicardipine were significantly so, indicated by red bars.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Knock-Out

Journal: Journal of Cellular and Molecular Medicine

Article Title: The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

doi: 10.1111/j.1582-4934.2008.00667.x

Figure Lengend Snippet: ( A ) This cartoon represents a possible distribution if Ca 2+ handling proteins related to caveolae and Cav-1 based on our findings. The peripheral ER/SR is shown near a caveola, with a restricted space in between them, containing a higher Ca 2+ concentration than in the general cytosol. Sequestered Ca 2+ is also shown associated with the caveolar extracellular membrane and adjacent raft tissue. This is the Ca 2+ which is protected from the Ca 2+ -free medium we used and provides ions for recycling to the restricted space and the ER/SR (and in Cav-1 knockout tissue provides some Ca 2+ for recycling). Owing to space limitations this recycling Ca 2+ is depicted adjacent to the restricted space. Proteins apparently co-localized with Cav-1 including VDCC, PMCA4, myogenic nNOS, and store operated Ca 2+ Channels are shown associated with the caveola membrane. Under the control conditions in Ca 2+ -free medium, the VDCC were the main entry route for Ca 2+ to be recycled, but store operated channels contribute when the VDCC are blocked. The SERCA pump and Leak or Release Sites in SR are also depicted. The Ca 2+ stored in the ER/SR is the other main source of Ca 2+ to maintain recycling and when it is emptied, recycling stops quickly. ( B ) This cartoon depicts the proposed changes in the Cav-1 knockout tissues. The restricted space is lost as well as the higher local Ca 2+ concentration which promotes its recycling to the ER/SR. VDCC, PMCA and store operated Ca 2+ Channels persist, but the myogenic nNOS is missing. The proximity of the remaining Ca 2+ molecules to the ER/SR is lost. There is also less sequestered Ca 2+ available for recycling. The result is more rapid loss of pacing in Ca 2+ -free medium. There is also less rapid clearing of Ca 2+ released or leaked from ER/SR by the PMCA and other clearance mechanisms. The main route for Ca 2+ entry remains the VDCC with a possible contribution from the store operated channel.

Article Snippet: For co-localization study of Cav-1 and store operated Ca 2+ channel (Orai-2) mouse anti-Cav-1 IgG (Cat. No. 160406, BD Transduction Labs; San Jose, CA, USA) and rabbit anti-Orai-2 IgG (Cat. No. ACC-061, Alomone Labs; Jerusalem, Israel) were used as primary antibodies.

Techniques: Concentration Assay, Knock-Out

Journal: The FASEB Journal

Article Title: Reduced myocyte complex N- glycosylation causes dilated cardiomyopathy

doi: 10.1096/fj.201801057R

Figure Lengend Snippet: A) Gel-shift immunoblot analyses indicate that the α2δ1 subunit of the cardiac Cav complex, but not the α1 subunit, is a target of GlcNAcT1. Sarcolemmal-enriched ventricular protein was treated with the endoglycosidases Endo-H, which removes high-mannose N-glycans but only in the absence of complex N-glycans, and PNGase-F, which cleaves all N-linked glycans. After treatment, protein was Western blotted and probed with antibodies specific for the α1 (top) and α2δ1 (bottom) subunits and compared with untreated lysates. Together, the data indicate that N-glycans from control sarcolemmal-enriched α2δ1 (bottom) exist almost entirely as complex N-glycans, whereas nearly all Mgat1KO α2δ1 subunits are in the high mannose N-glycan state. Additionally, the glycosidase studies suggest that there are few to no N-glycans of any type attached to α1 subunits. B) α2δ1 from control tissue possess high levels of sialic acids, as indicated by treatment with tissue prepared as described above with sialidase A, which cleaves all terminal sialic acids. α2δ1 from control tissue migrated ∼7 kDa farther into the gel after sialidase A treatment compared with Mgat1KO α2δ1. All data from glycosidase treatments represent results from 2 independent experiments and 2 animals from each group. MWs were calculated as described in the Materials and Methods and are listed in Table 3. C) α2δ1 protein expression is reduced in Mgat1KO ventricles. Top left: sarcolemmal-enriched ventricular protein from 3 control (lanes 1–3 from left) and 3 Mgat1KO (lanes 4–6 from left) hearts were Western blotted and probed with an α2δ1-specific polyclonal antibody. Bottom left: total protein/lane. Right: α2δ1 optical density from each sample was normalized to total protein from the corresponding lane. Ratios for each sample were compared to the mean control ratio value and indicate an ∼50% reduction in Mgat1KO α2δ1 protein expression (scatter plots indicate mean ± sem; n = 3; P = 0.003, Student’s t test).

Article Snippet: Protein was analyzed by Western blot on PVDF (1620177, Ca v α2δ1; Bio-Rad Laboratories) or nitrocellulose (10600003, Ca v α1; GE Healthcare Life Sciences, Little Chalfont St. Giles, United Kingdom), and immunodetection was performed as previously described using antibodies specific for Ca v α1 (ACC-003; Alomone Labs, Jerusalem, Israel) or Ca v α2δ1 (ACC-015; Alomone Labs) subunits.

Techniques: Gel Shift, Western Blot, Control, Glycoproteomics, Tissue, Expressing

Journal: The FASEB Journal

Article Title: Reduced myocyte complex N- glycosylation causes dilated cardiomyopathy

doi: 10.1096/fj.201801057R

Figure Lengend Snippet: Means ± sem calculated MWs in kilodaltons of Ca v subunits after glycosidase treatments

Article Snippet: Protein was analyzed by Western blot on PVDF (1620177, Ca v α2δ1; Bio-Rad Laboratories) or nitrocellulose (10600003, Ca v α1; GE Healthcare Life Sciences, Little Chalfont St. Giles, United Kingdom), and immunodetection was performed as previously described using antibodies specific for Ca v α1 (ACC-003; Alomone Labs, Jerusalem, Israel) or Ca v α2δ1 (ACC-015; Alomone Labs) subunits.

Techniques: Control