Anti hmgcr

Total protein samples were prepared from LNCaP and C4-2 cells treated with AIF or control by RIPA Lysis Buffer containing protease inhibitors. The amounts of proteins were determined by a PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). The equal amounts of the protein samples were loaded in SDS-PAGE gel for electrophoresis [26 (link)]. After electrophoresis, the separated proteins were transferred into polyvinylidene difluoride (PVDF) membranes and blocked by 5% reconstituted skim milk in PBS with Tween-20. Subsequently, the blotted membranes were incubated with primary antibodies for overnight. The detailed information of the used primary antibodies in this study as follow: anti-AR, anti-FASN (Santa Cruz Biotechnology, Dallas, TX, USA), anti-HMGCR (Abcam, Cambridge, UK), anti-BAX (Cell Signaling Technology, Danvers, MA, USA), anti-caspase-3 (Novus Biologicals, Centennial, CO, USA), anti-PARP, and anti-β-actin (GeneTex, Irvin, CA, USA). Next, these membranes were incubated with the secondary antibody. The visualization of the specific protein signal was detected by Enhanced Chemiluminescence Reagent kit (Amersham Biosciences, Arlington Heights, IL, USA) with the ImageQuantTM LAS 4000 system. Finally, the quantification of the protein band was analyzed by an ImageJ software (ij153-win-java8) with normalizing β-actin protein.

Dapagliflozin was purchased from the MedChemExpress Bio-Technology company (HY-10,450). A 10 mM stocking solution was dissolved and stored at -80˚C. RPMI 1640 medium and foetal bovine serum (FBS) were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Trypsin/EDTA was purchased from HyClone (Logan, UT, USA). WST-8 cell proliferation and cytotoxicity assay kit was purchased from Dojindo (CK04, Kumamoto, Japan). A cholesterol detection assay kit was purchased from Beijing Applygen Technologies (E-1015). The phalloidine staining reagent was purchased from the Sigma-Aldrich company. Nile acid was purchased from Macklin Inc (49,409). The siRNA specific for KLF-5 was designed and provided by the Genomeditech Company (Shanghai). Reagents for real-time PCR were purchased from Takara.
Primary antibodies are detailed as follows: Anti-ABCA1 (Abcam, Cambridge, MA, UK) for WB and IHC. Anti-KLF 5 (Abclonal, China) for WB and IHC. Anti-podocin (Abcam, UK); anti-nephrin (Abcam, UK); anti-LDLR (Abcam, UK); Anti-HMGCR (Abcam, UK); Anti-podocin (Abcam, UK); anti-Bax (Abcam, UK); anti-Bcl-2 (Abcam, UK); anti-caspase 3 (Abcam, UK); and, anti-GAPDH (Proteintech, China). Rats were purchased from the Animal Experimental Centre of Shandong University. All other materials and reagents were endotoxin-free and supplied by the central lab of Shandong Provincial Hospital.

MDA-MCF7 and MDA-MB231 cell lines were from Elabscience Biotechnology (Houston, Texas, USA), and MDA-MD453 was from ATCC (ATCC^® HTB-131, Manassas, VA, USA). Cell culture medium (Dulbecco’s modified minimum essential medium, DMEM), fetal bovine serum (FBS), penicillin G, streptomycin, glutamine, and sodium pyruvate were from GIBCO Invitrogen (Carlsbad, CA, USA). FBS lipid-depleted was from Biowest (Nuaillé, France). Human LDL was from Millipore (Burlington, Massachusetts, USA). Anti-nSMase3, anti-β-tubulin, anti-HMGCR and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Abcam (Cambridge, UK). ER antibody, clone 6F11, was from Leica Biosystems, Newcastle Upon Tyne, United Kingdom. PgR antibody, clone 16, was from Leica Biosystems, Newcastle Upon Tyne, United Kingdom. Ki-67 antibody clone MIB1 was from Agilent Dako, Glostrup, Denmark. 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2-tetrazoliumbromide (MTT), Chol standard, nSMase and were from Sigma-Aldrich (St. Louis, MO, USA). TaqMan SNP Genotyping Assay and Reverse Transcription kit were purchased from Applied Biosystems (Foster City, CA, USA). RNAqueous^®-4PCR kit was from Ambion Inc. (Austin, TX, USA). SDS-PAGE molecular weight standards were purchased from Nzythech (Lisboa, Portugal). Chemiluminescence kits were purchased from Amersham (Rainham, Essex, UK).

The antibodies used included anti-GAPDH (Bioword, AP0063), anti-NUCB2 (Proteintech, 26712-1-AP), anti-E-cadherin (Cell Signaling Technology, 24E10), anti-N-cadherin (Cell Signaling Technology, D4R1H), anti-Vimentin (Cell Signaling Technology, D21H3), anti-HisG (Cell Signaling Technology, D3I1O), anti-HMGCR (Abcam, ab242315), anti-SREBP2 (Abcam, ab30682), horseradish peroxidase (HRP) labeled sheep anti-Rabbit IgG (Cell Signaling Technology, 7074P2), and HRP labeled sheep anti-Mouse IgG-HRP (Sigma, A9917). The primary antibody titers for Western Blot or Immunohistochemistry are listed in Additional file 2: Table S1. Recombinant human purified Nesfatin-1 was purchased from Peprotech Company, and Rapamycin was obtained from Selleck Chemicals. Cholesterol was purchased from Beyotime, China.

Paraffin-embedded tissue sections of 4 μm thicknesses were deparaffinized with xylene, dehydrated in gradually diminishing concentrations of ethanol, and treated with 3% hydrogen peroxidase in methanol for 10 min to block endogenous peroxidase activity. The tissue sections were immersed in a 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using a 10 mM sodium citrate solution (pH 6.0). The sections were allowed to stay in the same solution while cooling for 20 min, and they were then rinsed in PBS. The sections were then incubated with a primary antibody (1:100 dilution) for 1 h at 37 °C. The primary antibody was follows: anti-HMGCR (Abcam, Cambridge, UK). The signal was visualized using an Envision System (DAKO, Carpinteria, CA, USA) for 30 min at 37 °C. 3,3′-diaminobenzidine tetrahydrochloride (DAB) was used as the coloring reagent, and hematoxylin was used as the counter-stain. The slides were examined with a slide scanner, Pannoramic MIDI, and analyzed with iSolution DT software.

Samples of liver tissue were washed three times with cold phosphate-buffered saline (PBS) before being lysed in radioimmunoprecipitation (RIPA) lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 1% NP-40; 0.1% sodium deoxycholate, 0.1% SDS, 150 mmol/L NaCl, and 1 mmol/L EDTA) supplemented with 1× protease and phosphatase inhibitor cocktail (Thermo, Fremont, CA, USA) on ice. The separated proteins were transferred onto a nitrocellulose membrane. The monoclonal anti-AMPK (1:100), anti-phospho-AMPK (1:200, Thr172), anti-HMGCR (1:100), anti-phospho-HMGCR (1:100, Ser872), anti-SREBP-2 (1:100), INSIG-1 (1:100), and anti-β-actin (1:3000) antibodies and secondary antibodies (1:10,000) were obtained from Abcam (Cambridge, MA, USA). Immunoreactivity protein bands were visualized using a ChemiDoc XRS+ System (Bio-Rad), and quantified with Gel Pro Analyzer software (Silk Scientific, Inc., Orem, UT, USA). The internal control, β-actin, was used to normalize differences due to loading variations.

Total protein was extracted by NP40 lysis buffer (Boster, Wuhan, China) and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated protein was transferred to polyvinylidene difluoride membranes and blocked with 5% bovine serum albumin (BSA) buffer. The BSA-blocked membranes were incubated separately with anti-SQLE (Proteintech, Wuhan, China), anti-LDLR (Proteintech, Wuhan, China), anti-HMGCR (Abcam, Cambridge, UK), anti-SREBP2 (Abcam, Cambridge, UK), anti-NPC2 (Proteintech, Wuhan, China), anti-CIMPR (Proteintech, Wuhan, China), and anti-Cas9 (Abcam, Cambridge, UK) antibodies and then incubated with secondary antibodies. The immunoblots were detected with ECL chemiluminescence substrate (Boster, Wuhan, China).