β actin c4 antibody

Adipose tissues were excised from ND-WT, ND-cKO, HFD-WT, and HFD-cKO mice, and homogenized in a radio-immunoprecipitation assay buffer with protease and phosphatase inhibitors (MilliporeSigma, St. Louis, MO, USA). The homogenized mixtures were centrifuged at 10,000× g for 10 min at 4 °C and each clear supernatant, excluding the upper layer of lipid, was transferred to a new tube. After additional centrifugation under the same conditions, protein lysates were prepared. Total protein concentration for protein lysates was measured using the Bio-Rad protein assay based on the Bradford dye-binding method (Bio-Rad, Hercules, CA, USA). The protein lysates were fractionated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes according to established procedures [19 (link)]. IL-8RB (K-19) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect CXCR2 protein levels. β-actin (C4) antibody (Santa Cruz Biotechnology) served as an internal loading control. The targeted protein bands were visualized by chemiluminescence detection kits (MilliporeSigma, St. Louis, MO, USA).

pMECs were cultured until 80~90% confluent and were then incubated with different concentrations of palmitate (0, 25, 50, 100, 200, 400, and 600 μM) for 24 h. After that, cells were collected and homogenized in RIPA lysis buffer (Beyotime, Nanjing, China) for assay of proteins related to lipogenic pathway. The homogenates were combined with equal volumes of SDS sample buffer, and the proteins were separated by electrophoresis on a 5~12% polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline with Tween, followed by overnight probing with the following primary antibodies: (1) CD36 (N-15) antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (2) ACACA (T-18) antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (3) DGAT1 antibody (1:500, Abcam, Cambridge, MA, USA), (4) SREBP1 (C-20) (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), (5) PPARγ (T-18) antibody (1:500, Abcam, Cambridge, MA, USA), and (6) β-actin (C4) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). β-actin was included as a loading (internal) control. After washing, membranes were incubated with secondary antibody (ABR, Golden, CO, USA). The chemiluminescent signal was detected by using ECL reagents (Beyotime, Nanjing, China), and bands were quantified by Image Processing Software (Image Pro Plus 6.0).

PMCs were seeded on 10 cm dish and incubated overnight and were then treated with HGF, for 3 days. After that, cells were lysis by cold RIPA buffer containing 1 × Protease inhibitor cocktail (ab20111, Abcam, Cambridge, MA, USA). The concentration of total protein levels was measured by a BCA protein assay kit (Pierce, Thermo Scientific, Waltham, MA, USA). 40 μg of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto to polyvinylidene difluoride (PVDF) membrane (Millipore) with a transfer apparatus (Bio-Rad, Hercules, CA, USA). PVDF membranes were blocked in 5% non-fat dry milk dissolved in TBS buffer and then incubated with primary antibodies including mouse p16INK4a (sc-166760, Santa Cruz, Santa Cruz, CA, USA) and β-Actin (C4) antibody (Santa Cruz sc47778, 1:500) overnight at 4 °C. The immunoblots were washed three times in TBS buffer for 10 min and then detected by the second antibody solution containing goat anti-mouse IgG-HRP (Santa Cruz) for p16INK4a, or goat anti-rabbit IgG-HRP (Santa Cruz) for β-Actin for 1 h in TBS buffer. The immunoblotted proteins were visualized using an enhanced Immobilon Western Chemiluminescent HRP Substrate Reagent (Millipore, Hayward, CA, USA) and detected by the Chemidoc XRS Chemiluminescent Gel Documentation Cabinet detection system (Bio-Rad).

GSTM1(1H4F2) (IHC and western): Novus Biologicals, LLC. (NBP2-22186); STAT3(C-20): Santa Cruz Biotechnology, Inc. (sc-482); Phospho-STAT3 (pSer727) Cell Signaling Technology, Inc. (#9134); p38 MAPK: Cell Signaling Technology, Inc. (#9212); Phospho-p38 MAPK (Thr180/Tyr182): Cell Signaling Technology, Inc. (#9211); NF-κB p65 (C22B4): Cell Signaling Technology, Inc. (#4764); NF-kB phospho-p65 (Ser536)(93HI): Cell Signaling Technology, Inc. (#3033); β-Actin Antibody (C4): Santa Cruz Biotechnology, Inc. (sc-47778).