Pro prep protein extraction kit

The effect of HFPS on the expressions of NF-κB, p-c-Jun, and MAPKs were assessed by Western blot analysis performed as described previously [43 (link),47 (link)]. In brief, cells were pretreated with HFPS and irradiated with UVB. After 1 h (for MAPKs assay) or 6 h (for NF-κB and p-c-Jun assay) incubation, cells were harvested. Proteins were extracted with the PRO-PREP protein extraction kit (iNtRON Biotechnology, Sungnam, Korea). The protein level of each sample was measured by a BCA™ kit. Total proteins (50 μg) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to pure nitrocellulose membranes. Membranes were blocked with 5% skim milk for 3 h at room temperature and incubated with primary antibodies overnight at 4 °C. After washing with TBS-T buffer, membranes were incubated with secondary antibodies for 3 h at room temperature. Finally, the protein bands were visualized using an ECL western blotting detection kit and exposed on X-ray films.

Total protein was extracted from samples of adipose tissue and isolated cells using a PRO-PREP Protein Extraction Kit (iNtRON Biotechnology, Kyungki-Do, Korea, 01434/40116). Aliquots containing 50 mg protein were separated by 8% SDS-PAGE. The primary antibodies were total or phosphorylated AKT (Ser473) (p-AKT), iNOS, Arg-1, PPARγ, total or phosphorylated STAT6 (Tyr641) (p-STAT6), and total or phosphorylated STAT3 (Tyr705) (p-STAT3). Total or phosphorylated AKT (Ser473) (p-AKT) (4685s and 4060s), total or phosphorylated STAT6 (Tyr641) (p-STAT6) (ab32520 and ab28829), and total or phosphorylated STAT3 (Tyr705) (p-STAT3) antibodies (12640S and 9145S) were from Cell Signaling Technology; iNOS (ab 15323) and Arg-1 (Ab60176) antibodies were from Abcam; and PPARγ antibodies (sc-7273) were from Santa Cruz Biotechnology. Goat anti-rabbit (ZB-2301), goat anti-mice (ZB-2305), and rabbit anti-goat IgG (ZB-2306) secondary antibodies were used. Blots were analyzed using ImageJ software (NIH, MD, USA).

As described previously (Kang et al. 2013 (link)), cells were trypsinized and lysed by Pro-Prep protein extraction kit (iNtRON Biotechnology, Seongnam, Korea). Equal amounts of protein extracts (20 &g) were separated by sodium dodecyl sulfate–polyacrylamide gel electro­phoresis and transferred to a nitrocellulose membrane (Invitrogen). Blots were blocked with 5% nonfat dry milk at room temperature. The blots were incubated with antibodies specific for angiopoietin-1 (ab183701, Abcam), angiopoietin-2 (ab155106, Abcam), phosphorylated Tie2 (Tyr992, #4221, Cell Signaling Technology), Tie2 (sc-9026), thrombospondin-1 (sc-59886) and &-actin (Santa Cruz Biotechnology) at specific dilution, followed by incubation with peroxidase-labeled secondary antibodies. Immunoreactive proteins were visualized using an enhanced chemiluminescence detection kit (Santa Cruz Biotechnology). Blot images were captured using ImageQuant LAS 4000 biomolecular imager (GE Healthcare Life Sciences). Blot intensities were quantified using ImageJ 1.48v software (http://imagej.nih.gov/ij) (Schneider et al. 2012 (link)).

The cells were irradiated with UVA and treated with PA at the indicated concentrations (0–2 μg/mL). Cells were washed with cold PBS twice and harvested. Next, proteins were extracted with the PRO-PREP protein extraction kit (iNtRON Biotechnology, Sungnam, Korea). The protein contents of total cell lysate were measured by using the BCA^TM protein assay kit (Sigma-Aldrich, St. Louis, MO, USA). The samples containing equal protein contents were loaded on 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and separated by electrophoresis, then transferred to a nitrocellulose membrane. At the blocking step, the membrane was blocked in 5% nonfat dry milk in TBST (25 mM Tris–HCl, 137 mM NaCl, 2.65 mM KCl, 0.05% Tween 20, pH 7.4) for 2 h. The 1:1000 dilutions of different primary antibodies in 5% nonfat dry milk solutions were individually incubated overnight at 4 °C. Thereafter, the membranes were washed and incubated with 1:3000 dilution secondary antibodies for 2 h at room temperature. All protein bands were developed using an ECL western blotting detection kit. The relative expressions of all proteins were analyzed by using the Image J program (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).