Horseradish peroxidase labeled secondary antibody

Western blot was performed as described previously [12 (link)]. The primary antibodies (Santa Cruz Biotechnology) were diluted as follows: TGF-β₁, 1:200; Smad7, 1:250; collagen I, 1:200; collagen III, 1:200; and β-actin, 1:500. Horseradish peroxidase–labeled secondary antibodies (Cell Signaling Technology) were diluted 1:1000 with 0.2 % TBS-T and 1 % skim milk. The protein bands on the Western blots were visualized using ECL Plus (Amersham, Arlington Heights, IL, USA). The relative band densities were normalized against β-actin.

Cell lysis followed by protein extraction was performed as described previously (Basu et al., 2014). Lysates were subjected to immunoblotting with antibodies specific for ETS‐1, ETS‐2, MMP‐9, MMP‐13, HIF‐1α, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), glyceraldehyde‐3 phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; 1:2000); MMP‐2, protein kinase B (AKT), p‐AKT, proliferating cell nuclear antigen, E‐cadherin, N‐cadherin and vimentine (Cell Signaling, Danvers, MA, USA; 1 : 1000). Bands were visualized by reacting horseradish peroxidase‐labeled secondary antibodies (Cell signaling; 1 : 5000) with the ECL substrate (BioRad) by chemiluminesence.

Gigantol was extracted from stems of Dendrobium draconis Rchb.f., as previously described [45 (link)] and dissolved in dimethylsulfoxide (DMSO) at the indicated working concentrations. 3-(4,5-Dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), Hoechst 33342, propidium iodide (PI), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), cocktail protease inhibitor, hematoxylin, and eosin were purchased from Sigma chemical, Inc. (Chemical Express, Bangkok, Thailand). RPMI-1640 medium, DMEM, phosphate buffer saline (PBS), glutamine, penicillin, and streptomycin were purchased from Gibco company (Gibthai, Bangkok, Thailand). Primary antibodies against CD133, ALDH1A1, total AKT, phosphorylated AKT (Ser473), total STAT3, phosphorylated STAT3 (Ser727), and GAPDH, horseradish peroxidase labeled secondary antibodies, and RIPA lysis buffer were purchase from Cell Signaling Technology (Theera Trading, Bangkok, Thailand). Pentobarbital sodium injection was purchased from Ceva Sante Animal (VET AGRITECH, Nonthaburi, Thailand). 3,3′-Diaminobenzidine tetrahydrochloride hydrate was purchased from TCI Co., LTD (Chemical Express, Bangkok, Thailand). Primary antibodies of Ki-67 and α-SMA and matched secondary antibodies were purchased from DAKO (Medicare Supply, Bangkok, Thailand).

Cells were transfected with 50 nM negative control RNA mimics (denoted as NC), mimics-miR-29a/b, anti-scramble (control anti-miRNA) and anti-miR-29a/b siRNA duplexes for SPARC and COL3A1 (Gene Pharma) in 24-well plates. Cell samples and nuclear/cytoplasmic extracts were prepared according to the manufacturer’s instructions (Thermo), collected 48 h later, and analyzed using Western blotting. GAPDH (Cell Signaling Technology) was used as a loading control. Protein expression was detected by incubation with either rabbit polyclonal anti-SPARC or anti-COLA1 (Santa Cruz Biotechnology). Immunoreactive bands were detected by ECL (Amersham, USA) using horseradish peroxidase-labeled secondary antibodies (Cell Signaling Technology).

RAW 264.7 cells were seeded in a 6-well plate, pretreated with 4-hydroxycinnamaldehyde (12.5 µM and 25 µM) and ECC (200 µg/mL) for 2 h, and then treated with LPS at 100 ng/mL. The cells were lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.25% deoxycholate, 1% NP-40, and 1 mM EDTA), and the protein concentration was determined using BCA reagent (Thermo Fischer Scientific, Rockford, IL, USA) according to the manufacturer’s instructions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). After the transfer, the membranes were blocked with 5% nonfat dried milk/TBST (20 mM Tris pH 7.4, 150 mM NaCl, and 0.2% Tween-20) for 1 h and then incubated overnight at 4 °C with a primary antibody from Cell Signaling Technology (Danvers, MA, USA). After washing with TBST, the membranes were probed with horseradish peroxidase-labeled secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Finally, the proteins were visualized using enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, Pittsburgh, PA, USA). The densities were normalized to the expression of GAPDH or the total amount of each protein.