Bradford reagent

For triglyceride measurements, ten adults were homogenized in 500 μL PBS containing 0.2% Triton X, heated at 70 °C for 5 min, and centrifuged at 18,400 × g for 10 min. Ten microliters of supernatant was used to measure the TAG level by Serum Triglyceride Determination Kits (TR0100-1KT, Sigma). Protein amounts were measured using Bradford Reagent (P0006, Beyotime, Shanghai, China). TAG level was normalized to the protein amount.
For glucose measurements, ten adult flies were homogenized in 200 μL distilled water after being weighed and centrifuged at room temperature at 8000 × g for 10 min. The supernatant was used to measure the glucose level using Glucose Detection Kits (BC2505, Solarbio, Beijing, China). For trehalose measurements, ten adult flies were weighed and homogenized in 200 μL extracting solution. After standing at room temperature for 45 min, extractions were centrifuged at room temperature at 8000 × g for 10 min. The supernatant was used to measure the trehalose level using Trehalose Detection Kits (BC0335, Solarbio). The glucose and trehalose levels were normalized to the weight. Each genotype was measured three times.

Cells samples were treated with lysis buffer (Beyotime, China) and mice xenograft tissues were treated with RIPA (Beyotime, China) and sonication. Total protein concentrations were detected using BCA or Bradford reagent (Beyotime, China). Each protein sample (30 μg) was separated on a 12% gel (Bio-Rad, Amercia), transferred to nitrocellulose membranes (Immobilon-P, Millipore, Bedford, MA, USA). The membranes were incubated in 5% milk in Tris-buffered saline with Tween20 for 2 h at room temperature to block nonspecific antibody binding, and subsequently incubated with the anti-phosphorylated-NF-κB(p65) (p-NF-κB(p65)) (1:1000), anti-Bim (1:1000), or anti-β-actin (1:5000) antibodies overnight at 4 °C. The membranes were washed with Tris-buffered saline with Tween20 and incubated with the peroxidase-conjugated goat anti-rabbit (1:5000) or anti-mouse (1:5000) antibodies for 2 h at room temperature. Bound antibodies were observed using the enhanced chemiluminescence immunoblotting detection reagent (Thermo Scientific, IL, USA).

After the treatment, the cells were lysed using an RIPA lysis buffer and the protein content was determined using Bradford reagent (Beyotime Biotechnology, Shanghai, China). Afterwards, Western blotting was performed. The samples were separated using 10% (w/v) sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis to separate the proteins. Electrophoresis was performed at 80 V for 2 h. The proteins were transferred to PVDF membranes at 100 V for 1 h. Afterwards, the membrane was blocked with 5% (w/v) non-fat dry milk at room temperature for 2 h, the corresponding antibody was added and incubated overnight at 4◦C, and it was incubated with the secondary antibody for 2 h at room temperature. Finally, the proteins were visualized using ECL reagents (YEASEN, Shanghai, China) and a gel imager (Syntyos, Cambridge, MA, USA).

The open reading frames (ORFs) of Sm4CL were amplified with pET primer pairs (Table S1), and subcloned into a pET32a vector. After the sequences were confirmed, each construct was transformed into Escherichia coli strain BL21 (DE3) for heterologous expression. The transgenic cultures were incubated at 37 °C until the OD₆₀₀ reached 0.4–0.6, then the recombinant proteins were induced at 16 °C for 16 h after adding 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG). N-terminal hexahistidine-tagged proteins were purified by passing through a Ni-NTA Sefinose His-bind column (Bio Basic Inc., Markham, ON, Canada), and then were exchanged through an Ultrafiltration tube (Millipore, Billerica, MA, USA) in the presence of binding buffer (20 mM Tris–HCl, 500 mM NaCl, pH 8.0). Protein concentrations were determined with the Bradford reagent (Beyotime, Shanghai, China), using BSA as a standard. The resulting purified proteins were monitored on SDS-PAGE, using Coomassie Blue R250 staining.  The migration of standard molecular weight markers (10–170 kDa) (Fermentas, Waltham, MA, USA) was used to estimate the molecular masses of the target proteins.

Changes in intracellular osmotic pressure were determined by measuring glycerol content using the Glycerol Assay Kit (Jiancheng, Nanjing, China), according to the provided protocol. Protein concentrations were determined using the Bradford reagent (Beyotime, Shanghai, China) with BSA employed as a standard. The concentrations of glycerol were calculated as the values of the content of glycerol divided by the content of protein. Results are the mean of triplicate experiments ± SDs.

BV2 cells were lysed in RIPA buffer and determined with Bradford reagent (Beyotime, cat. no. P0006C). The protein samples were separated on 10% tris-glycine polyacrylamide gels (Sangon Biotech, China, cat. no. C681102). Transfer proteins to nitrocellulose membranes with the Transfer-Blot Turbo Transfer System (Bio-Rad, USA). Membranes were blocked with Protein-Free Rapid Blocking Buffer (Epizyme Biotech, China, cat. no. PS108P) for 15 min and incubated overnight with primary antibodies. The following primary antibodies were used: anti-tubulin antibody (1:1,000; ABclonal, China, cat. no. AC026) and anti-IL-1β (1:1,000; RD, USA, cat. no. AF-401). Tubulin was detected using goat anti-rabbit secondary antibody (1:5,000; Jackson Immuno Research Labs, USA, cat. no. 111-035-003), and IL-1β was detected using donkey anti-goat secondary antibody (1:2,000; Beyotime, China, cat. no. A0181). Signals were visualized with the ChemiDoc Imaging System (Bio-rad).