Gbox chemi xt4 gel imaging system

Liver tissue was homogenized in RIPA lysis buffer containing protease inhibitor (Complete mini EASY pack, Roche, Basel, Swiss) to extract protein. The concentration was determined by BCA kit (CoWin Bioscience, Beijing, China) and protein was resolved with 10% polyacrylamide gel, then transferred to the PVDF membrane (Millipore, Billerica, MA, USA). After blocking, they were incubated overnight in antibody dilutions of GRP78 (1:1000) (CST, Boston, MA, USA, 3183), P-PERK (1:1000) (CST, Boston, MA, USA, 3179), P-EIF2α (1:1000) (CST, Boston, MA, USA, 3398), P-NFκB (1:500) (CST, Boston, MA, USA, 3033), P-IREα (Abcam, Cambridge, UK, ab148187), TRAF2 (1:1000) (Abcam, Cambridge, UK, ab126758) or β-actin (1:1000) (Huaan biological technology, Hangzhou, China), and secondary antibody were incubated for 2 h at room temperature. The images of blots were acquired by GBOX Chemi XT4 gel imaging system (Syngene, Cambridge, UK).

Brains, cortex, and hippocampi of sham and TBI mice were dissected on ice and immediately processed. Tissues were homogenized in RIPA buffer (10 mM Tris–HCl, Triton X-100 0.5%, 1% NP-40, 5 mM EDTA, 1% sodium deoxycholate, and 1% SDS) supplemented with protease and phosphatase inhibitor mixture (Protease: Amresco, VWR Life Science; Phosphatase: 25 mM NaF, 100 mM Na₃VO₄ and 30 µM Na₄P₂O₇) using a Potter homogenizer and then passing through a tuberculin syringe. Samples were centrifuged at 14,000 rpm at 4 °C for 10 min. Protein concentration was determined using a BCA protein assay kit (Pierce, ThermoFisher Scientific, USA). Samples were resolved by SDS-PAGE and transferred to PVDF membranes. Western blot was performed as previously described [21 (link)] with overnight incubation of primary antibodies at 4 °C. Blots were developed using a chemiluminescence detection kit (West Pico, ThermoFisher, USA). Images were obtained with a G:BOX Chemi XT4 Gel imaging system (Syngene). Membranes were stripped for 30 min at room temperature using a harsh stripping buffer (6 M GnHCl, 0.2% NP-40, 100 mM β-mercaptoethanol, 20 mM Tris–HCl, pH 7.5) and washed thoroughly 4 times with PBS containing 0.1% Tween-20. After stripping, membranes were tested again with different antibodies where needed.

The hippocampus of mice from all experimental groups was dissected on ice and immediately processed as previously described [19 (link)]. The mouse hippocampal samples were homogenized in RIPA buffer (10 mM Tris-Cl, pH 7.4, 5 mM EDTA, 1% NP-40, 1% sodium deoxycholate, and 1% SDS), supplemented with inhibitors of proteases (Amnesco, VWR Life Science, Suwanee, GA, USA) and phosphatases (25 mM NaF, 100 mM Na₃VO₄ and 30 µM Na₄P₂0₇), using a Potter homogenizer. The samples were centrifuged twice at 14,000 rpm at 4 °C for 15 min. The protein concentration is determined using a BCA protein assay kit (Pierce). Samples were resolved by SDS-PAGE, followed by immunoblotting onto PVDF membranes. Immunodetection was performed with their respective primary antibodies incubated overnight at 4 °C followed by detection with secondary antibodies coupled to peroxidase and revealed using a chemiluminescence detection kit (West Pico, ThermoFisher, Waltham, MA, USA). The images were obtained with a G: BOX Chemi XT4 Gel Imaging system (Syngene, Bangalore, India).