Western Blot Analysis of Neuroinflammatory Markers

Rats were quickly decapitated under deep anaesthesia, and the left hemispheres were isolated for Western blot as described before [32 (link)]. The polyvinylidene fluoride (PVDF) membranes were incubated with primary antibodies as follows: TXNIP (1:500; Abcam, Cambridge, Mass), TRX1 (1:2000; Abcam), NLRP3 (1:1000; Abcam), cleaved Caspase-1 (1:500; Abcam), cleaved IL-1β (1:500; Abcam), cleaved Caspase-3 (1:1000; CST, Danvers, MA), BCL-2 (1:1000; CST), sXBP1 (1:100; Santa Cruz), phosphorylated-PERK (p-PERK, 1:200; Santa Cruz), eukaryotic translation initiation factor-2α (eIF2α) (1:100; Santa Cruz), phosphorylated-eIF2α (p-eIF2α, 1:500; Abcam), carbohydrate response element-binding protein (ChREBP) (1:100; Santa Cruz), activating transcription factor 5 (ATF-5) (1:2000; Abcam) and β-actin (NeoBioscience Technology Co., Ltd, Beijing, China). Membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies, visualized with the enhanced chemiluminescent reagent kit (ECL, Engreen Biosystem Co., Ltd. Beijing, China) and analysed using the Fusion system (Fusion fx 7 Spectra, Vilber, France). Results are expressed as a percentage of the values for β-actin.

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Zhao Q., Che X., Zhang H., Fan P., Tan G., Liu L., Jiang D., Zhao J., Xiang X., Liang Y., Sun X, & He Z. (2017). Thioredoxin-interacting protein links endoplasmic reticulum stress to inflammatory brain injury and apoptosis after subarachnoid haemorrhage. Journal of Neuroinflammation, 14, 104.

Publication 2017

TXNIP NLRP3 cleaved Caspase-1 cleaved IL-1β sXBP1 phosphorylated-PERK (p-PERK, phosphorylated-eIF2α (p-eIF2α, ChREBP) β-actin (ECL,

Actin Activating transcription factor Anaesthesia Antibodies Bcl 2 Carbohydrate binding protein Caspase 1 Caspase 3 Eukaryotic translation initiation factor Horseradish peroxidase Il 1β Membranes Polyvinylidene fluoride Rats Response element Transcription factor 5 Trx1 Txnip Western blot

Corresponding Organization :

Other organizations : First Affiliated Hospital of Chongqing Medical University, Chongqing Medical University, West China Hospital of Sichuan University, Sichuan University

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Variable analysis

independent variables

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dependent variables

Levels of the following proteins as measured by Western blot: TXNIP, TRX1, NLRP3, cleaved Caspase-1, cleaved IL-1β, cleaved Caspase-3, BCL-2, sXBP1, phosphorylated-PERK (p-PERK), eukaryotic translation initiation factor-2α (eIF2α), phosphorylated-eIF2α (p-eIF2α), carbohydrate response element-binding protein (ChREBP), and activating transcription factor 5 (ATF-5)

control variables

None explicitly mentioned

positive controls

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negative controls

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