Pierce ecl system

Whole-cell lysates from approximately 2 × 10⁵ cells per each sample were separated on 15% SDS-PAGE gels and transferred to a nitrocellulose membrane. Immunoblots were all blocked with 5% nonfat dry milk in Tris-buffered saline, 0.1% (TBS-T) prior to incubation with primary antibodies. The Anti-poly (ADP-ribose) polymer (1:1000 Abcam, ab14459) was stained overnight at 4 °C. For PARP1 and DNMT1 protein analyses, equivalent amount of whole-cell lysates were separated on 7% SDS-PAGE gels and transferred to a nitrocellulose membrane. Immunoblots were stained overnight with the following primary antibodies: Anti-PARP1 (1:1000 Active motif, Carlsbad, CA, USA; 39559), Anti-DNMT1 (1:1000, Abcam, Cambdrige, UK; ab19905). All secondary horseradish peroxidase (HRP)-conjugated antibodies were diluted 1:5000 and incubated for 1 h at room temperature with TBST/5% milk. Immuno-reactive proteins were detected using the Pierce^® ECL system (Thermo Scientific, Whaltman, MA, USA; #32106).

Cells were incubated at 30°C for 16 h in the presence (1 μg/mL) or absence of FLC. Cells were washed with sterile water once and then were subjected to cell lysis using the Bead Ruptor12 system (OMNI International, USA) in lysis buffer (PBS [pH 7.2 to 7.6]) containing 5 mM EDTA (pH 8.0), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1.0% protease inhibitor cocktail (Sigma). Proteins were separated by 4 to 20% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking, anti-GFP antibody (Santa Cruz) or antitubulin antibody (Abbkine) was used for probing GFP-tagged proteins or tubulin, which were then detected using the secondary antibody goat anti-mouse IgG conjugated with horseradish peroxidase (HRP) (Santa Cruz) and the Pierce ECL system (Thermo Fisher Scientific, MA, USA).

Total proteins were extracted from the tissues by homogenization in RIPA buffer containing protease inhibitors. The homogenate was cleared by centrifugation at 4 °C for 30 min at 12 000 rpm, and the supernatant (containing the protein fraction) was collected. Protein concentration in the supernatant was measured using the BCA Protein Assay Kit (Beyotime). A total of 20 mg protein per sample was resolved in a 10–12% SDS‐PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked with 5% w/v BSA in tris‐buffered saline containing 0.2% Tween‐20, and then incubated at 4 °C overnight with primary antibody. The membranes were then washed and probed with the appropriate horseradish peroxidase‒conjugated secondary antibody and detected using the Pierce ECL System (Thermo Scientific). See Table S2, Supporting Information, for the antibodies used.

Frozen renal tissue was homogenized, protein samples were prepared as described [57 (link)] and separated on a denaturing SDS-PAGE gel [58 (link)]. After electrophoresis, the gels were electroblotted onto PVDF membranes (Hybond-P, GE Amersham, Munich, Germany), blocked with Rotiblock (Roth, Karlsruhe, Germany) for 1 h and incubated overnight with a primary antibody to chemerin. Protein bands were visualized with secondary horseradish peroxidase-conjugated IgG antibodies (Santa Cruz Biotechnology, 1:50,000), using the Pierce ECL+ system (Thermo Fisher Scientific, Waltham, MA, USA). Blots were quantified using a luminescent imager (LAS-1000, Fujifilm, Berlin, Germany) and Aida 2.1 image analysis software (Raytest, Berlin, Germany). Loading of the blot was quantified by Amido Black staining solution (Sigma, Taufkirchen, Germany).