Radioimmunoprecipitation assay buffer

An RNeasy Plus kit (Qiagen, Germantown, MD, USA), a high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA), and a Power SYBR Green PCR master mix kit (Applied Biosystems) were employed with listed PCR primers (Table 1). For detecting GFP-labeled 4T1.2 cells in the right femur, total DNA was isolated with QIAamp DNA mini kit (Qiagen) for qPCR.
For Western blotting, cells were lysed by a radio-immunoprecipitation assay buffer (Santa Cruz). Proteins were fractionated by 10-15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Antibodies against ATF4, caspase 3, cathepsin K, eIF2α, p-eIF2α (Ser51), LC3A/B II, NFATc1, Chk1, p-Chk1 (Ser296) (Cell Signaling, Danvers, MA, USA), TRAP (Abcam, Cambridge, MA, USA), and β-actin (Sigma) were utilized.

HUVECs treated with 20μM DHA were collected at different time points. To generate a positive control for p38 MAPK activation, a group of HUVECs were treated with 1 μg/ml anisomycin for 1 h. Cell lysates were prepared in radioimmunoprecipitation assay buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 50 mM NaF, 1% NP-40, 0.1% deoxycholate, 0.1% SDS and 1 mM EDTA] (Santa Cruz Biotechnology, Inc.), supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin (Santa Cruz Biotechnology, Inc.). Cleared cell lysates were subjected to SDS-PAGE using 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 2.5% non-fat milk, and incubated with primary antibodies at 4°C overnight in phosphate-buffered saline Tween-20 (Santa Cruz Biotechnology, Inc.). The primary antibodies used were total-p38, phospho-p38 (Cell Signaling Technology, Inc.) and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Immunoreactivity was visualized with horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence reagent (Santa Cruz Biotechnology, Inc.). The blots were analyzed using a Bio-Rad imaging system (Bio-Rad, Hercules, CA, USA).

Total protein from cells was extracted by lysis in radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology, #sc-24948) with protease inhibitor cocktail (Cell Signaling Technology, #5872). Protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, #23227). Samples with equal amounts of protein were fractionated on SDS–polyacrylamide gels (Bio-Rad, #4561084), transferred to polyvinylidene difluoride (Millipore Sigma, #IPVH00005) membranes, and blocked in 5% skim milk (Cell Signaling Technology, #9999s) in TBST (0.1% Tween 20 in tris-buffered saline) for 1.5 h at room temperature. The membranes were then incubated at 4 °C overnight with primary antibodies [phospho-NF-κB p65 1:1000, Cell Signaling Technology, #3033; NF-κB p65 1:1000, Cell Signaling Technology, #8242; mSPARCL1 1:500, R&D systems, #AF2836-SP; β-actin 1:2000, Cell Signaling Technology, #4970]. After the membranes were washed with TBST, incubations with 1:4000 dilutions (v/v) of the secondary antibodies were conducted for 2 h at room temperature. Protein expression was detected using the ChemiDoc XRS+ System (Bio-Rad). β-actin was used as a loading control.

Total cell lysates were obtained using a radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology). Protein concentration was determined using a Bradford protein assay (Bio-Rad) against a bovine serum albumin standard. A total of 30 to 50 μg of total protein per sample was run on a 12% SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane. Primary antibodies against MIPS (Invitrogen #PA5-44105, 1:500 dilution), p-AMPK (Cell Signaling #2531S, 1:1000 dilution), AMPK (Cell Signaling #2793S, 1:1000 dilution), and IP6K1 (GeneTex # GTX103949, 1:5000 dilution) were used. Corresponding HRP-tagged secondary antibodies (Invitrogen) or Alexa Fluor 488-labeled fluorescent secondary antibodies (Thermo Fisher) were used. The signal was detected using an iBright FL1500 imaging system (Thermo Fisher). iBright analysis software was used for band intensity quantification. No-stain protein labeling reagent (Invitrogen, A44717) was used to normalize for protein loading.

The whole-cell lysates were obtained from HL-60R cells using radioimmunoprecipitation assay buffer (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and 25 µg protein was subjected to 10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham, Pharmacia Biotech, Milan, Italy) using a semi-dry fast blot apparatus (Bio-Rad, Milan, Italy). Membranes were blocked with 5% (w/v) BSA in PBS-0.1% (v/v) Tween 20 for 1 h and then filters were incubated with primary antibodies raised against GAPDH (1:20,000; Sigma-Aldrich Srl, Milan, Italy.), XIAP (1:500; Cell Signaling Technology, Inc. Danvers, MA, USA), survivin (1:2000, Abcam Limited, Cambridge, UK), Bcl-2 ( 1:1000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) and P-gp (1:100, Invitrogen, Milan, Italy). The hybridization was visualized using an enhanced chemiluminescence detection kit (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific Life Technologies Italia, Monza, Italy) and the Versa DOC imaging system (BioRad Laboratories, Milan, Italy). Immunoblots were quantified by densitometry and results were expressed as arbitrary units (protein/GADPH).