Anti fabp4

Western blotting was performed using the following primary antibodies: anti-CLUH (A301-764A; Bethyl Laboratories, Montgomery, TX), anti-PPAR-γ (SC-7273; Santa Cruz Biotechnology, Santa Cruz, CA), anti-FABP4 (#2120; Cell Signaling Technology, Beverly, MA), anti-Flag (F3165, Sigma-Aldrich) and anti-GAPDH (SC-25778, Santa Cruz Biotechnology). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) reagents (Santa Cruz Biotechnology).

3T3-L1 cells were purchased from the American Type Culture Collection (USA). 3T3-L1 cells were maintained in DMEM (Thermo Fisher Scientific, USA), bovine calf serum (BCS, HyClone, USA) and 1% penicillin/streptomycin (Corning Inc., USA). MDA-MB-231 and MTV/TM-011 cells were obtained from Korean Cell Line Bank (Republic of Korea). MDA-MB-231 and MTV/TM-011 cells were cultured in RPMI (Corning Inc.) containing 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Corning Inc.). Cells were maintained at 37 °C in a humidified atmosphere with 5% CO₂/95% air. Recombinant mouse GREM2 protein was obtained from R&D Systems (USA). Recombinant mouse IL-6 protein was purchased from Sino Biological Inc. (China). Rabbit polyclonal GREM2 antibody was purchased from Abcam (UK). Anti-PPARγ, anti-C/EBPα, anti-FABP4, anti-vimentin, anti-slug, anti-twist1, and anti-β-actin were obtained from Cell Signaling Technology (USA). Epigallocatechin gallate (EGCG), metformin, and PKH26/PKH67 fluorescent cell linker kits were purchased from Sigma-Aldrich (USA).

Western blot was performed according to standard procedure. The treated cells were lysed with ice‐cold RIPA buffer containing both protease and phosphatase inhibitors. The cytoplasmic and nuclear extracts from cells were separated using nuclear and cytoplasmic Extraction kits (#78833; Thermo Fisher Scientific). Protein concentrations were quantified using a BCA protein assay kit (Thermo Fisher Scientific). Primary antibodies including anti‐β‐actin (#4790), anti‐FABP4, anti‐CD68, anti‐PCNA (#13110), anti‐p21 (#2974), anti‐Cyclin B1 (#12231), anti‐NF‐κB/p65 (#6956), anti‐Histone H3 (#4499), anti‐FLAG tag (#8146), anti‐ubiquitin antibodies (#3933), and FAK antibody sample kit (#9330) were all purchased from cell signaling technology (Beverly, USA). Anti‐March5 antibody was purchased from ProteinTech (#12213‐1‐AP). Anti‐human USP30 antibody was purchased from Abcam (#ab235299). All bands were visualized by enhanced chemiluminescence and quantified by Image J analysis software.

Protein was extracted from cells as previously described (51 (link)). For Western blotting analysis, 30 to 40 μg of protein was resolved by 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (Bio-Rad), incubated overnight at 4 °C with specific primary antibodies in blocking buffer (Tris-buffered saline, 0.1% Tween-20, and 5% milk or bovine serum albumin), and detected with alkaline phosphatase (AP)–conjugated secondary antibody. The antibodies used were anti-NPRC (1:2000; Novus Biologicals; #NBP1-31365), anti-PPARγ (1:2000; Cell Signaling Technology; #2435), anti-FABP4 (1:1000; Cell Signaling Technology; #2120), anti-ADIPOQ (1:1000; Cell Signaling Technology; #2789), anti-GAPDH (1:2000; Protein Tech; #10494-1-AP), and anti-rabbit IgG-AP (1:20,000; Sigma; #A3687).

Protein expression was evaluated by western blot. Frozen mouse epididymal fat tissue was homogenized in liquid nitrogen. Tissue was lysed in RIPA lysis buffer (Sigma) containing 1% PI. Cell lysate was kept on ice for 1 h and subsequently centrifuged at 13,000 rpm for 20 min at 4°C. Total proteins (30 μg protein/test) were isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (DC, Invitrogen). The membrane was blocked with 5% skim milk and subsequently incubated at 4°C with the following primary antibodies (1:5000 dilution): anti-AMPK, anti-SREBP-1c, anti-FAS, anti-ACC, anti-PPARγ, anti-C/EBPα, anti-FABP4, and anti-adiponectin (Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was incubated with IgG HRP-conjugated secondary antibody (1:2000) for 2 h at room temperature. Ponceau S was used for staining the protein bands. β-actin was used as the loading control. Proteins were visualized using enhanced-chemiluminescence (ECL) location reagent and quantified with the ImageJ program.

OC cells (20,000/cm² in 35 mm or 60 mm dishes, Corning, Corning, NY, USA) were allowed to adhere overnight and were cultured for 48 or 72 h in media with 5% FBS ± inhibitor. After lysis, cellular proteins were subjected to SDS–PAGE, blotted, and immunostained as previously described [35 (link),36 (link)] using anti-FABP4 (1:200, #2120, Cell Signaling Technology, Boston, MA, USA), anti-FABP5 (1:500, #39926, Cell Signaling Technology), anti-CD36 (1:1000, #14347, Cell Signaling Technology), anti-ARF6 (1:500, #3546, Cell Signaling Technology), anti-FABP6 (1:250, #96122, Abcam, Cambridge, UK), anti-LDLR (1: 1000, #52818, Abcam), anti-FATP2 (1:500, # 175373, Abcam), and anti-actin (1:1000, #1616, Santa Cruz Biotechnology, Dallas, TX, USA). Secondary antibodies were peroxidase-labeled donkey anti-rabbit (1:15,000, #16284, Abcam), donkey anti-goat IgG (1:15,000, #2020, Santa Cruz Biotechnology), donkey anti-mouse (1:10,000, #715-035-150, Jackson ImmunoResearch, West Grove, PA, USA), or donkey anti-sheep (1:10,000, #A16041, Thermo Fisher, Waltham, MA, USA). Detection was by enhanced chemiluminescence.