T0004

HEK 293T cells transfected with 2.5, 3.5 and 5 µg of pcDNA3.1-CD147 were lysed 24h and 48h post-transfection with 1x RIPA buffer (abcam) and 1x Complete EDTA free protease inhibitor cocktail (Roche). Cell lysates were quantified by DC protein Assay (Bio-Rad) following manufacturer’s instructions and then mixed with 4x Laemmli’s sample buffer (Bio-Rad) with 10% β-mercaptoethanol. Protein extracts were loaded and run on 10% SDS-PAGE gels and transferred onto a PVDF membrane (Bio-Rad). Membranes were blocked with 5% skimmed milk/TBST 1X (TBS1X-0.1% Tween 20) for 1 h and then incubated overnight at 4°C with primary antibodies; rabbit anti-CD147 (abcam ab108308; 1:1,000 dilution) and mouse anti-GAPDH (Affinity Biosciences, T0004; 1:10,000 dilution) in blocking solution. Following washing, membranes were incubated with the respective secondary antibodies for 1 h at room temperature; goat anti-rabbit IgG (IRDye^® 800CW) (ab216773) and goat anti-mouse IgG (IRDye^® 680RD) (ab216776) diluted 1:20,000. After washing, the fluorescence signal was visualized using an Odyssey CLx Imaging System (LI-COR Biosciences).

Western blot was conducted and analysed as previously described.
⁹ (link) Proteins (50 μg) were separated by 8%, 10% and 12% SDS‐PAGE, respectively, according to molecular weight. The primary antibodies used were as following: GAPDH (1:1000, T0004, Affinity Biosciences, China), kidney injury molecule 1 (KIM‐1, 1:1000, AF1817, R&D Systems, USA), α‐SMA (1:1000, AF1032, Affinity Biosciences, China), FN (1:1000, ab2413, Abcam, UK), COL I (1:1000, ab138492, Abcam, UK), PAR4 (1:1000, AF5371, Affinity Biosciences, China). The secondary antibodies used were as following: goat anti‐mouse (1:2000, L3032, Signalway Antibody Co., China) and goat anti‐Rabbit (1:2000, L3012, Signalway Antibody Co., China).

Total proteins were extracted using tissue protein extraction reagents. Total tissue proteins (20 µg/lane) were loaded and separated on sodium dodecyl sulfate polyacrylamide gels electrophoresis (SDS-PAGE) at 120 V for 90 min, followed by transferring to polyvinylidene fluoride (PVDF) membranes (Millipore, USA) at 300 mA for 2 h. After that, Membranes were incubated in 5% (w/v) milk for 1 h followed by incubation overnight at 4 °C with primary antibodies, including, rabbit anti-TREM2 (1:1000, PAB37053, Bioswamp, China), rabbit anti-TREM2 (1:1000, PA5-87933, ThermoFisher, USA), rabbit anti-MBP (1:1000, 78896S, Cell Signaling Technology, USA), Acvr2B (1:1000, ab128544 and ab180185, Abcam, UK), mouse anti-GAPDH (1:1000, T0004, Affinity, USA), rabbit anti-β-actin (1:1000, AF7018, Affinity, USA) overnight at 4 °C. After washes, the membranes were incubated with corresponding secondary antibodies for 1 h. The ECL western blotting detection kit (P007, ABP Biosciences, Japan) and an enhanced chemiluminescence system were used to examine protein bands. ImageJ software was used to analyze the gray value of protein bands.

MCF-7 and T47D cells were lysed in lysis buffer (#KGP250/KGP2100, Keygen Biotech, Nanjing, China) containing protease and phosphatase inhibitors following the manufacturer’s protocol. Proteins were subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto a PVDF membrane, then incubated with CCNE2 (#ab40890, Abcam; dilution rates of 1:3000), CDCA5 (#67418-1-Ig, Proteintech; dilution rates of 1:1000), RAD51(#14961-1-AP, Proteintech; dilution rates of 1:1000), MCM10 (#12251-1-AP, Proteintech; dilution rates of 1:500), ERα(#ab108398, Abcam; dilution rates of 1:2000), PR(#ab206926, Abcam; dilution rates of 1:1000), Vinculin(#26520-1-AP, Proteintech; dilution rates of 1:2000) and GAPDH antibodies (#T0004, Affinity; dilution rates of 1:5000) at 4 °C overnight, respectively. The next day, the membranes were exposed to secondary antibodies (#S0101, #S0100, Lablead, Beijing, China; 1:5000 dilution) at room temperature for 1 h. Protein bands were measured using Syngene GeneGenius (SYNGENE, GeneGnome XRQ NPC, Cambridge, UK.).

For the determination of GLUT4, Akt, p-Akt, DMRT2, and FXR protein levels, cells or tissues were lysed in RIPA buffer with 1% PMSF for protein extraction following the methods described before. For membrane protein isolation, the Membrane and Cytosol Protein Extraction Kit (Beyotime, China) was used. A bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Waltham, MA, USA) was used for the detection of protein concentration. The protein sample (50 μg) was fractionated using an SDS-polyacrylamide gel (10%–15%) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes. Then, the membranes were probed with proper primary antibodies listed as follows: GLUT4 (66846-1-1G, Proteintech), Akt (ab8805, Abcam), p-Akt (ab38449, Abcam), DMRT2 (PA5-41694, Thermo), FXR (25055-1-AP, Proteintech), UCP-1 (23673-1-AP, Proteintech), BSEP (18990-1-AP, Proteintech), SHP (NR0B2, Boster), Cyp7A1 (D161909-0025, Sangon Biotech), GAPDH (T0004, Affinity Biosciences), and α-tubulin (11224-1-AP, Proteintech). Then, anti-mouse or anti-rabbit IgG coupled to peroxidase was used as a secondary antibody (Beyotime, China). Immunoreactivity was detected using the Odyssey Infrared Imaging System (Gene Company Limited, Hong Kong, China). The band intensity was measured by ImageJ software. The protein expression was normalized to α-tubulin or GAPDH.

Total protein was extracted from tissues or cells using RIPA lysis buffer (CW2333, CWBIO, China) containing proteinase and phosphatase inhibitors. Nuclear and cytoplasmic protein of TM3 cells was extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Western blot analysis was conducted as previously described [35 (link)]. The primary antibodies included anti-FOXO4 (ab128908, 1:1000; Abcam, USA), anti-p53 (ab26, 1:200; Abcam, USA), anti- Ser15-phospho-p53 (ab1431, 1:500; Abcam, USA), anti-p21 (ab188224, 1:1000; Abcam, USA), anti-p16 (MAB2416, 1:500; Abnova, USA), anti-3β-HSD (sc-515120, 1:200; Santa Cruz, USA), anti-CYP11A1 (GTX56293, 1:500; Gene Tex, USA), anti-CYP17A1 (ab125022, 1:1000; Abcam, USA), anti-IL-1α (16764-1-AP, 1:500; Proteintech, USA), anti-IL-1β (ab9722, 0.2 μg/ml; Abcam, USA), anti-IL-6 (ab9324, 0.4 μg/ml; Abcam, USA), anti-IL-10 (DF6894, 1:500; Affinity, USA), anti-TNF-α (17590-1-AP, 1:500; Proteintech, USA), anti-TGF-β (18978-1-AP, 1:500; Proteintech, USA) and anti-GAPDH (T0004, 1:5000; Affinity, USA) antibodies. GAPDH was used as the control.

Drugs were added to the BV2 microglia as previously described. Twelve hours after administration, cells were collected and cleaved; protein concentration was quantified; protein lysate (25 μL) was electrophoresed and transferred onto PVDF membrane (pore size: 0.45 μm). After the transmembrane was completed, the PVDF membrane was sealed with 5% skim milk for 2 h and incubated with the specified primary antibody—against P-AMPK (T55608S, 1:1000, Abmart, Shanghai, China), mTOR(T55306S, 1:5000, Abmart), P-mTOR (T56571S, 1:5000, Abmart), LC3-II/LCI (T55992F, 1:500, Abmart), IL-1β (TA5103, 1:500, Abmart), P62 (T55546S, 1:5000, Abmart), NLRP3 (15101s, 1:1000, Cell Signaling Technology (CST), Danvers, MA, USA), GAPDH (T0004, 1:1000, Affinity, Cincinnati, OH, USA), caspase-1 p20 (AF4005, 1:1000, Affinity) or AMPK (AF6423, 1:1000, Affinity)—at 4 °C overnight. PVDF membrane and HRP-labeled secondary antibody were cultured at room temperature for 1 h and rinsed 3 times. Visualization of the protein bands was achieved with an enhanced chemiluminescence (ECL) kit (P0018FS, Vazyme, Nanjing, China) and imaging system (3500R, Tanon, Shanghai, China), whose grayscale values were calculated using ImageJ version 5.0.