Enhanced chemi luminescence (ecl)

Preadipocytes were collected when which grew by roughly 1 × 10⁷ in vitro culture and washed with phosphate buffer solution. Then, the cells were added into the total protein extract, which contained less than 1 ml of protease inhibitor (SinoGene, Beijing). The protein concentration in the lysate was calculated by the Bradford protein assay reagent (SinoGene, Beijing). The protein products were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with the following primary antibodies: anti-CaMKK2 and anti-β-actin (Abcom, England). Subsequently, the membranes were immunoblotted with the secondary goat anti-rabbit antibody after incubating overnight with primary antibodies. The immunoreactivity was visualized by enhanced chemiluminescence (ECL, Engreen).

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.