Ibright cl1500 imaging system

FLAG-tagged SREBP1a was overexpressed in HEK293 cells, and the protein was purified using anti-FLAG agarose beads (Sigma). DNA probes corresponding to wild type or the E-box deletion of the cyclin D1 promoter were prepared by PCR and purified. The following primers were used: forward, 5’ CCC CGT CCT TGC ATG CTA AAT 3’; reverse, 5’ CCA AAG AAT CTC AGC GAC TGC A 3’. The template was the pGL3Basic-cyclin D1 promoter construct mentioned above. The 10× binding buffer contains 200 mM Tris–HCl pH 7.5 and 500 mM NaCl. The final reaction mixture contains 2 μl 10× binding buffer, 20% glycerol, 1 μg sheared salmon sperm DNA, 1 mM MgCl₂, 1 mM dithiothreitol (DTT), and 0.5 μg bovine serum albumin (BSA). Where indicated, 500 ng of probes and 4 μl of purified FLAG-SREBP1a were added to the reactions. To generate a supershift, anti-FLAG antibody (M5) was used. The electrophoresis mobility shift assay (EMSA) reactions were separated on 4% polyacrylamide gels with 0.5× TBE buffer, stained with SYBR Safe, and visualized on an iBright CL1500 Imaging System (Invitrogen).

Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on Bolt NuPAGE 4–12% Bis-Tris Plus gels (Invitrogen, ThermoFisher Scientific; #NW04122BOX) using Bolt NuPAGE MOPS SDS running buffer and transferred to PVDF blotting membranes (Amersham Hybond) with Trans-Blot SD Semi-Dry Transfer Cell. The following antibodies were used: Phospho-IRF-3 (Ser396) (4D4G) Rabbit mAb (Cell Signaling, #4947); IRF-3 (D614C) Rabbit mAb (Cell Signaling, #11904); EiF4G (C45A4) Rabbit mAb (Cell Signaling, #2469) and β-Actin (8H10D10) antibody (Cell Signaling, #3700). HRP-coupled anti-mouse (NA9310V, GE Healthcare) and anti-rabbit IgG, HRP-linked antibody (Cell Signaling, #7074) were used as secondary antibodies. Peroxidase activity was visualized with SuperSignal West Pico PLUS chemiluminescent substrate (#A34580, ThermoFisher Scientific) or SuperSignal West Atto chemiluminescent substrate (#A38556, ThermoFisher Scientific) with the Invitrogen iBright CL1500 Imaging System.

Dissected striata from saline or Olaparib-Wt and R6/2 half brains were homogenized with the RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail (Sigma Aldrich, USA) and centrifuged at 13,000 × g for 20 min. Equal amounts of protein were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes, and incubated with rabbit NLRP3, (1:1000 Abcam, Novus Biologicals), mouse caspase-1 (1:1000, Abcam, Novus Biologicals, Italy), rabbit pAKT, AKT (1:1000 Cell Signaling Technology), rabbit pCREB, CREB (1:1000 Immunological Sciences, Italy), rabbit pERK, ERK (1:1000 Immunological Sciences, Italy), mouse PSD95 (1:1000, Abcam Novus Biologicals, Italy), rabbit caspase-8 (1:1000, Abcam, Novus Biologicals, Italy), rabbit PARP-1 (1:1000, Abcam, Italy), and mouse GAPDH (1:10,000; Sigma Aldrich, St Louis, MO, USA) antibodies, overnight at 4 °C. After being washed with Tris-buffer saline (TBS)/Tween 20, membranes were incubated with HRP-labeled secondary antibodies. Protein signals were visualized using the Invitrogen iBright CL 1500 Imaging system.

Transcript levels for the mouse and human genes of F2rl1 (PAR2), Mif (MIF), pref-1 (PREF1), PPARγ (PPARG), ATGB1, ATGA5, Tnfa (TNF), Il6 (IL6) and Il1b (IL1B) (Tables S1 and S2), etc were measured by qPCR. The primer sequences have been listed in Tables S1 and S2. Briefly, RNAs were extracted by TRIzol™ Reagent (Invitrogen) and iScript cDNA Synthesis Kit (Bio-Rad) was used to synthesize complementary DNA. SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad) was performed to measure gene expression levels in the QuantStudio 6 Flex System (Applied Biosystems). The delta–delta Ct method was used to quantify gene expressions normalized by Gapdh.
Phosphorylation and total levels of ERK, AMPK and ACC, the contents of MIF and p115 in adipose tissue or cells, and the released levels of p115 and MIF in cell culture medium were evaluated by Western blot. Protein extracts were performed in a lysis buffer containing cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (ROCHE). According to the manufacturer’s instructions, protein concentrations were examined using Bio-Rad Protein Assay Dye Reagent (Bio-Rad). Protein quantification was performed by using an iBright™ CL1500 Imaging System (Invitrogen).

pWAT and BAT western blot: Tissues were minced, and lysed in RIPA buffer (50 mM Tris HCl pH 8, 150 mM NaCl, 1%NP-40, 0.5% SDS) containing protease inhibitors and Calyculin A (Cell Signaling) using a pellet disruptor. Protein lysates were quantify using the BCA assay (Thermo Fisher Scientist), and were normalized for total protein concentration. Total protein was subjected to SDS-PAGE, transferred to PVDF membranes (Millipore), incubated with blocking solution (5% BSA in TBS pH 7.4) for 1 h and incubated overnight at 4 °C in primary antibody: Adipoq (1:1000, 2789 T, Cell Signaling), Fabp4 (1:1000, 2120 S, Cell Signaling), Atgl (1:1000, 2439 S, Cell Signaling), P-Hsl (Ser563) (1:1000, 4139 T, Cell Signaling), Hsl (1:1000, 18381T, Cell Signaling), Hsp90 (1:5000, 610419, BD Biosciences), and UCP1 (1:20000, 14670S, Cell Signaling). After secondary incubation at room temperature for 1 h, protein bands were visualized using Clarity Western ECL substrate (BioRad) using the iBright CL1500 Imaging system (Invitrogen). Quantification of western blots was done by using ImageJ software. A representative experiment is shown, n = 5. Full western blot membranes are showed in Supplementary Fig. 16A-B.

Total proteins from specified cells extracted using RIPA lysis buffer contains 1X protease and phosphatase inhibitor. Protein concentration of each sample measured using BCA protein assay kit (ThermoFisher, #23227) as per the manufacturer’s instructions. Then, equal concentration of proteins from each sample were taken and mixed appropriate volume of 5X protein sample buffer supplemented with reducing agent. Subsequent incubation at 98°C for 10 min, equal amount of each protein sample was subjected to SDS-polyacrylamide gel electrophoresis using 4–12% gel and transferred to polyvinylidene difluoride (PVDF) membrane (Bio-Rad, #162–0177). The protein transferred membranes were immunoblotted with appropriate primary antibodies for overnight at 4°C followed by appropriate horseradish peroxidase (HRP) conjugated secondary antibodies for 1 h at room temperature. Signal was visualized by enhanced chemiluminescence substrate (ThermoFisher, #F32106) and exposed using iBright CL1500 imaging system (Invitrogen). Band intensities were quantified using ImageJ software and normalized to β-actin, heatmaps were generated using GraphPad Prism 9.5.1.

To screen angiogenic-related factors in both FDM and sFDM, the Proteome Profiler™ human angiogenesis array kit (ARY007; R&D Systems, Minneapolis, MN, USA) with an array of 55 angiogenesis-related proteins was employed according to the manufacturer’s instructions. Briefly, a nitrocellulose membrane was blocked with a supplied block buffer, then added with a mixture of FDM lysates and a cocktail of biotinylated detection antibodies. Incubated overnight at 4 °C, the membrane was rinsed with a wash buffer, and streptavidin-horseradish and chemiluminescent detection reagents were then added sequentially. Once chemiluminescence was carried out via iBright CL1500 imaging system (Invitrogen, Waltham, MA, USA), the collected data were quantitatively analyzed using iBright analysis software 4.0.1.