We analyzed intramuscular creatine concentrations in autopsied skeletal muscle specimens derived from five subjects who had SBMA (age: mean ± standard deviation [SD] = 74.0 ± 5.3 years), five subjects who had ALS (67.8 ± 3.2 years), and five subjects who had other diseases (73.8 ± 6.1 years), including progressive supranuclear palsy (n = 2), Parkinson's disease (n = 1), Lewy body dementia (n = 1), and multiple system atrophy (n = 1). There were no statistically significant differences in patient's age at the time of examination among SBMA, ALS, and disease controls (DCs). Neither were there significant differences in the postmortem intervals from death to autopsy among the groups (SBMA, 512.6 ± 374.8 min; ALS, 648.4 ± 494.9 min; and DC, 403.2 ± 339.5 min [mean ± SD]). Individual 100 mg muscle samples were homogenized in 1 mL of CelLytic lysis buffer (Sigma‐Aldrich, St. Louis, MO) containing a protease inhibitor cocktail (Thermo Scientific, Waltham, MA). The samples were then centrifuged at 2500g for 15 min. The resultant supernatant was used for analyzing the concentrations of creatine using an enzymatic method at LSI Medience Co. ( Tokyo, Japan). Moreover, using the same method, we also analyzed intramuscular creatine concentrations in skeletal muscle specimens derived from the wild‐type mice (n = 8) and AR‐97Q mice (n = 8), a transgenic mouse model of SBMA.7
Cellytic lysis buffer
Manufactured by Merck Group
CelLytic lysis buffer is a ready-to-use solution designed for the efficient lysis of eukaryotic cells. It is formulated to facilitate the extraction and solubilization of proteins from various cell types.
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Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Hrp linked anti rabbit igg, by Cell Signaling Technology (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Jak2 mab, by Cell Signaling Technology (1 mentions)
Phospho jak2 mab, by Cell Signaling Technology (1 mentions)
Stat1 mab, by Cell Signaling Technology (1 mentions)
Phospho stat1 mab, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protein g agarose beads, by Roche (1 mentions)
Anti myc tag antibody, by Cell Signaling Technology (1 mentions)
Anti flag tag antibody, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Ab178401, by Abcam (1 mentions)
Anti myc agarose, by Merck Group (1 mentions)
Myc mouse mab, by Cell Signaling Technology (1 mentions)
7 protocols using cellytic lysis buffer
1
Creatine Levels in Neurodegenerative Diseases
2
Western Blot Analysis of Signaling Pathways
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Cell were lysed in CelLytic lysis buffer (Sigma-Aldrich) with a protease inhibitor cocktail (Sigma-Aldrich) and a phosphatase inhibitor cocktail (Sigma-Aldrich). The protein concentration of cell lysates was measured using a BCA protein assay (Thermo Scientific). Five micrograms of protein was separated on 4–12% NuPage Bis-Tris gels (Invitrogen) and transferred to a polyvinylidene fluoride (PVDF) membrane using the iBlot Dry Blotting System (Invitrogen), according to the manufacturer's protocol. Blots were blocked in PBS (−) with 0.05% Tween 20 (Sigma-Aldrich) buffer (PBST) with 2% nonfat dry milk, followed by incubation overnight in PBST with 2% nonfat dry milk and primary antibodies. The following were used as primary antibodies and purchased from Cell Signalling Technology: JAK2 mAb, phospho-JAK2 mAb, STAT1 mAb, phospho-STAT1 mAb, p44/42 MAPK (Erk1/2) mAb, phospho-p44/42 MAPK (Erk1/2) mAb, and β-actin antibody. After washing in PBST, membranes were incubated with the HRP-linked anti-rabbit IgG (Cell Signalling Technology). Blots were visualized by ECL Prime (Amersham Pharmacia, Uppsala, Sweden) according to the manufacturer's protocol and with a film processor (Konica SRX-101A; JZ Imaging & Consulting, Willoughby, OH, USA).
3
Isolation of Cardiac Mitochondria
4
ADAMTS1 and APP Interaction Analysis
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HEK 293T cells co-transfected with ADAMTS1-FLAG and APP-Myc were washed with PBS buffer and solubilized in ice-bath by CelLytic lysis buffer (C2978, Sigma-Aldrich, Shanghai, China) containing protease inhibitors. Clear lysates were incubated with primary antibodies overnight at 4°C and then with protein G Agarose beads (11243233001, Roche, Basel, Switzerland) for 2 h at 4°C. After five-time rinse with TBS buffer, immunoprecipitated proteins were recovered from the beads by boiling for 5 min in sample buffer, and then analyzed by immunoblotting.
Antibodies were diluted with TBST buffer containing 5% skim milk and listed below: anti-FLAG tag antibody (1:5,000,RRID:AB_262044 , F1804, Sigma-Aldrich, Burlington, United States), anti-Myc tag antibody (1:5,000, RRID:AB_331783 , 2276, Cell Signaling Technology, Danvers, United States) and anti-Na+/K+ ATPase antibody (1:5,000, RRID:AB_2227873 , 14418-1-AP, Proteintech, Wuhan, China).
Antibodies were diluted with TBST buffer containing 5% skim milk and listed below: anti-FLAG tag antibody (1:5,000,
5
Western Blot Analysis of NS5A Protein
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Cells were lysed in CelLytic™ lysis buffer (Sigma-Aldrich) added of protease inhibitors (Sigma-Aldrich). Ten micrograms of protein were resolved by SDS/PAGE and transferred to a PVDF membrane. Membranes were blocked in 10% (w/v) dried skimmed milk powder in Tris-buffered saline with 0.1% Tween-20 (TBS-T). Membranes were probed with anti-NS5A IgG (Macdonald et al., 2003) or mouse anti-GAPDH IgG (AbCam) in 5% (w/v) dried skimmed milk in TBS-T. The antibodies were detected with the secondary horseradish peroxidase-conjugated antibody and in-house enhanced chemiluminescent reagent.
6
Measuring Reverse CrAT Activity in C2C12 Myotubes
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Reverse CrAT activity was measured using C2C12 myotubes. Naïve mitochondrial pellet isolated from the cells was re-suspended in CelLytic lysis buffer (Sigma Aldrich) and sonicated at five times one second pulses on ice. Two hundred µg of the mitochondrial lysate was warmed at 37°C for 5 min, and then was added to pre-incubated (37°C, 5 min) 50 mM Tris-HCl buffer (pH 7.8) containing 500 µM AcCT, 500 µM CoA and 1 mM EDTA.(25 (link)) The mixture (total volume of 200 µl) was incubated at 37°C for 0, 5, 10, 15 and 20 min, and AcCoA and CT were quantified by LC-MS/MS. Productions of AcCoA and CT were calculated per mg mitochondrial protein.
7
Immunoprecipitation of Protein Complexes
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Cells or brain tissues washed once with cold PBS were solubilized or homogenized on ice in Cellytic lysis buffer (C2978; Sigma-Aldrich) supplemented with protease inhibitors (250 mM PMSF, 5 mg/ml pepstatin A, 10 mg/ml leupeptin, and 5 mg/ml aprotinin). Cleared lysates were obtained by centrifugation at 12,000 rpm for 10 min at 4°C, and 0.7–1.2 mg of the lysates were used for immunoprecipitation. Lysates were incubated with primary antibodies overnight at 4°C and incubated for 2 h with a 50% slurry of protein A/G Sepharose. After three rinses with PBS, immunoprecipitated proteins were recovered from the beads by boiling for 10 min in sample buffer or by competition with the tag peptide and then analyzed by immunoblotting. Anti-FLAG mouse mAb (#8146; Cell Signaling Technology), Myc mouse mAb (#2276; Cell Signaling Technology), and anti-Myc agarose (A7470; Sigma-Aldrich) were used against epitope tags. For endogenous coimmunoprecipitation from primary cultured astrocytes, cell lysates were incubated with rabbit anti–GLT-1 antibody (ab178401; Abcam). Anti-EAAT2 antibody (ab205247; Abcam) was used for in vivo coimmunoprecipitation from mouse hippocampal tissues, which were dissected from the hippocampus ipsilateral to KA injection at 5 wk after SE.
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