α spectrin

Manufactured by Merck Group

α-spectrin is a key structural protein component of the cytoskeleton in eukaryotic cells. It is involved in maintaining the stability and shape of the cell membrane.

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Spectrin (5 citations)

4 protocols using α spectrin

Neuroprotective Signaling Pathway Assessment

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WB was performed with cortical tissue from sham-operated and the ipsilateral side of HI-injured animals. The following primary antibodies were used: NR2B (BD Transduction Laboratories, San Jose, CA), phospho-Y1252 NR2B and phospho-Y1336 NR2B (PhosphoSolutions, Aurora, CO), phospho-Y1472 NR2B and phospho-Y1070 NR2B (Cell Signaling Technology, Boston, MA), α-spectrin (Millipore, Billerica, MA), cleaved-caspase 3 (Cell Signaling), p47^phox (Millipore), gp91^phox (BD) and β-actin (Santa Cruz Biotechnology). Appropriate secondary HRP-conjugated antibodies were used and signal was visualized with enhanced chemiluminescence. Image J was used to measure the optical densities (OD) of blots on radiographic film after scanning.

Knox R., Brennan-Minnella A.M., Lu F., Yang D., Nakazawa T., Yamamoto T., Swanson R.A., Ferriero D.M, & Jiang X. (2014). NR2B phosphorylation at Tyrosine 1472 contributes to brain injury in a rodent model of neonatal hypoxia-ischemia. Stroke; a journal of cerebral circulation, 45(10), 3040-3047.

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Protein Extraction and Western Blot Analysis

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After treatments, slices were collected in dry ice and homogenized in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton) containing protease and phosphatase inhibitor cocktail (Thermo). Protein concentrations were measured using the BCA protein assay kit (Thermo). Equal amounts of proteins (20–30 μg) were processed for SDS-PAGE and western blot, as previously described63 . The primary antibodies used were PTEN (1:1000, Cell signaling), phospho-ERK T202/Y204 (1:2000, Cell Signaling), ERK (1:3000, Cell signaling), phospho-Akt S473 (1:1000, Cell signaling), Akt (1:2000, Cell signaling), phospho-S6 S240/S244 and S6 (both 1:1000, Cell signaling), phospho-eIF2α S51 (1:500, Cell signaling), eIF2α (1:1000, Cell signaling), PP2A B56 alpha subunit (1:200, Santa Cruz), GLUA1 N-terminal (1:200, Santa Cruz), GLUA1 C-terminal (1:1000, Millipore), α-spectrin (1:2000, Millipore), PHLPP1 (1:1000, Millipore), actin (1:10000, Millipore).

Zhu G., Briz V., Seinfeld J., Liu Y., Bi X, & Baudry M. (2017). Calpain-1 deletion impairs mGluR-dependent LTD and fear memory extinction. Scientific Reports, 7, 42788.

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Western Blot Analysis of Signaling Pathways

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Western blot analyses were carried out as previously described.19, 20 The following primary antibodies were used for immunoblotting: phospho‐ERK, STIM1 and GAPDH obtained from CST (Cell Signaling Technology), ERK and calpain obtained from Santa Cruz Biotechnology, Orai1 and β‐actin obtained from Sigma‐Aldrich, α‐spectrin obtained from Millipore, and EP4 obtained from Cayman.

Osawa K., Umemura M., Nakakaji R., Tanaka R., Islam R.M., Nagasako A., Fujita T., Yokoyama U., Koizumi T., Mitsudo K, & Ishikawa Y. (2019). Prostaglandin E2 receptor EP4 regulates cell migration through Orai1. Cancer Science, 111(1), 160-174.

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Protein Expression Analysis of Abdominal Aortic Tissue

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Abdominal aortic tissue lysates were extracted in radioimmunoprecipitation assay lysis buffer, and protein content was measured using a Bradford assay (Bio-Rad, Hercules, CA). Protein extracts (20–30 μg) were resolved by SDS-PAGE (6.0 or 7.5 % wt/vol) and transferred electrophoretically to PVDF membranes (Millipore). After blocking with non-dry fat milk (5 % wt/vol), membranes were probed with primary antibodies. The following antibodies were used (please see Major Resources Table in the Data Supplement): calpain-1 domain IV (Abcam, catalog No: ab39170), calpain-2 (Abcam, catalog No: ab39165), filamin A (Abcam, catalog No: ab76289), talin (Abcam, catalog No: ab71333), α-spectrin (Millipore, catalog No: MAB1622), ILK-1 (Cell Signaling, catalog No:3862), FAK (Cell Signaling, catalog No:3009), vinculin (Abcam, catalog No: ab73412), paxillin (Abcam, catalog No: ab32264), collagen (Abcam, catalog No: ab34710), elastin (Abcam, catalog No: ab217356), calnexin (Enzo, catalog No: ADI-SPA-860-F) and β-actin (Sigma-Aldrich, catalog No: A5441). Membranes were incubated with appropriate HRP-labeled secondary antibodies. Immune complexes were visualized by chemiluminescence (Pierce, Rockford, IL) and quantified using a Kodak Imager.

Muniappan L., Okuyama M., Javidan A., Thiagarajan D., Jiang W., Moorleghen J.J., Yang L., Balakrishnan A., Howatt D.A., Uchida H.A., Saido T.C, & Subramanian V. (2021). Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice. Arteriosclerosis, thrombosis, and vascular biology, 41(5), 1694-1709.

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