β actin 13e5

THP-1 monocytes were seeded in a 6-well plate with a cell density of 2,100,000 cells/mL and differentiated into macrophages by adding 200 nM of PMA to the cell culture medium and culturing them for 72 h. After differentiation, cells were exposed to 50% DPBS, 10% (v/v) AGEs, and 3 ng/mL LPS for 24 h. After incubation, cells were lysed with a Triton buffer consisting of 150 mM sodium chloride, 0.1% Triton-X, and 50 mM Tris pH 8, and the protein content was assessed with the BCA Assay (Thermo Scientific, Waltham, USA) according to the manufacturer’s protocol.
A sample of 10 µg with reducing laemmli buffer was loaded onto a 10% mini protean TGX precast gel (Bio-rad, Hercules, CA, Verenigde Staten) and run in a Bio-rad cell. Protein was transferred to an Immobilon-P PVDF Membrane (Merck, Darmstadt, Germany). Membranes were analyzed using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The primary antibodies used were Glucocorticoid Receptor (D6H2L), Phospho-Glucocorticoid Receptor (Ser211), and β-actin (13E5) from Cell Signaling Technology (Danvers, MA, USA) and Anti-phospho-Glucocorticoid receptor Ser226 from Millipore (Temecula, USA). The secondary antibody used was Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology).

A lysis buffer (150 mM NaCl, 0.1% SDS, 0.02% NaN₃, 1% NP-40, and 50 mM pH 8.0 Tris) containing cocktail of inhibitor for protease and phenylmethylsulfonyl fluoride (1 mM) was used to lysate cells. Western blotting assays were performed with Biolad Protean II minigel system. Then, 50 μg protein sample was injected in every gel (12%) well of, and then shifted to PVDF membrane after electrophoresised. 1× TBST (5% dry skimmed milk) was used to dispute the non-specific binding. Then the membranes were sequentially incubated with primary and secondary antibody, respectively. Furthermore, these membranes were determined with enhanced ECL (chemiluminescence) reagent of Hyperfilm ECL kit, and exposed using x-ray film.
The primary antibodies included α-SMA (CST 19245, dilution 1: 1200) and Smad 3 (C67H9) (CST 9523, dilution 1: 1500), p-Smad 3 (CST-9520, dilution 1: 1000), TGF-β (56E4) (CST-3709, dilution 1: 1200), SIRT3 (CST2627, dilution 1: 1000), E-cadherin (24E19) (CST 3195, dilution 1: 800), β-actin (13E5) (CST 4970S, dilution 1: 4000) (Cell Signaling, Danvers, MA, USA).

Raw sweet corn (Zea mays L. ssp. saccharata Sturt) was purchased from Talaad Thai, the main wholesale market (Pathum Thani Province, Thailand). Recombinant Human IL-1β (carrier-free) and enzyme-linked immunosorbent assay (ELISA) kits were purchased from BioLegend Way (San Diego, CA, USA). Specific antibodies raised against phospho-p44/42 (Erk1/2) (20G11) (#4376), p44/42 (Erk1/2) (#9102), phospho-SAPK/JNK (81E11) (#4668), SAPK/JNK (#9258), phospho-p38 (D3F9) XP^® (#4511), p38 (#9212), phospho-NF-kappaB p65 (Ser536) (93H1) (#3033), NF-kappaB p65 (C22B4) (#4764), iNOS (D6B6S) (#13120), CD54/ICAM-1 (E3Q9N) XP^® (#67836), β-actin (13E5) (#4967), and anti-rabbit IgG HRP-linked Antibody (#7074) for western blot were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Enhanced chemiluminescence (ECL) detection kits were acquired from Thermo Scientific (Rockford, IL, USA). Precision Plus Protein™ Kaleidoscope™ prestained protein standards were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA, USA).

ResolvinD1 (17(S)-RvD1) was purchased from Cayman Chemical Company (USA). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were from Beyotime Corporation (Shanghai, China). Bronchial epithelial cell growth medium (BEGM, CC-3170) was purchased from Lonza (USA). Antibodies to E-cadherin (24E10), Phospho-NF-kB p65 (Ser536) (93H1), histone H3 (D1H2), NF-kB p65 (D14E12), and β-actin (13E5) were from Cell Signaling Technology (MA, USA), antibodies to ZO-1 (ab96587), HO-1 (ab13248), and Nrf2 (ab137550) were purchased from Abcam (USA), IKBα (WL01936) and p-IKBα (WL02495) were from Wanleibio Company (Shenyang, China). The transfection reagents for Nrf2 were from Hanbio (Shanghai, China).

Total protein was extracted from NSCLC tumor tissues and cells using RIPA lysis buffer containing protease inhibitor cocktail (Invitrogen, USA), then quantified by a BCA Protein assay kit (Solarbio, Beijing, China). 10 ug protein was used for SDS-PAGE electrophoresis. Separated protein in SDS_PAGE gel was transferred onto the PVDF membrane (BioRed, USA). After blocking with 5% skimmed milk for 1 h, the membrane was then incubated with primary antibodies: FGF11 (1:1000; Cell Signaling Technologies #3139, MA, USA), HIF-1α (1:1000, Cell Signaling Technologies #3716), β-Actin (13E5) (1:2000, Cell Signaling Technologies #4970) and GAPDH (1:2000; Cell Signaling Technologies #2118) for 2 h or overnight at 4℃. The membrane was washed 3 times with TBST for 5 min each. After wash, the membrane was further incubated with HRP-linked secondary antibody (1:3000; Cell signaling #7074, MA, USA) at room temperature for 1 h. Then the membrane was washed 4 times with 1 × TBST and the protein bands were visualized using an enhanced chemiluminescence kit (Santa Cruz, TX, USA) and photographed on a gel imager system (Bio-Rad).