Total RNA from the brain or liver was extracted using Trizol Reagent (Life Technologies) and treated with RNA-free DNAase I to remove remnant DNA as described previously (Kim et al. 2014 (link)). cDNA used for gene detection was synthesized as described previously using SuperScript III reverse transcriptase and random primers (Kim et al. 2014 (link)). The primers used for this study were purchased from Fisher Scientific as below: UBXN6, Mm01272178; GRP78, Mm00517691; UBE2J2, Mm01263784; PDIA6, Mm01270904; ERLEC1, Mm00458735; HYOU1, Mm00491279; Manf, Mm00512511; 18s rRNA, Mm03928990. The relative expression was normalized to internal control (18s rRNA) using cycle time (Ct). The relative difference between the control and treatment group was expressed and calculated as relative increases using 2−ΔΔCt and setting control as 1.
Grp78
Manufactured by Thermo Fisher Scientific
Sourced in Ireland
GRP78 is a molecular chaperone protein that plays a crucial role in the folding and processing of proteins within the endoplasmic reticulum (ER) of cells. It is involved in the unfolded protein response, a cellular stress response mechanism that helps maintain ER homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Phospho eif2α, by Cell Signaling Technology (1 mentions)
Total eif2α, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Spliced xbp1, by BioLegend (1 mentions)
Western lightening plus ecl, by PerkinElmer (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
2 protocols using grp78
1
Quantitative Analysis of Brain and Liver mRNA
2
Western Blot Analysis of UPR Markers
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Western blotting procedures has been described previously.48 (link) The primary antibodies used were ATF6 (Abcam, Cambridge, UK, Cat# ab122897), spliced XBP1 (Biolegend, London, UK, Cat# 619502), PERK (Cell signalling, Cat# C33E10), GRP78 (Fisher Scientific Ireland Ltd, Cat# PA1-014A), phospho-eIF2α (Cell signalling, Cat# 9721), total eIF2α (Cell signalling, Cat# 9722) and or β-actin (Sigma, Cat# A-5060) overnight at 4 °C. The membrane was washed three times with PBS-0.05% Tween and further incubated in appropriate horseradish peroxidase-conjugated secondary antibody (Fisher Scientific Ireland Ltd) for 90 min. Signals were detected using Western Lightening Plus ECL (PERKin Elmer, Dublin, Ireland).
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