Phosphorylated p38

Equal amounts of lysates were resolved by sodium dodecyl-polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The blots were visualized using an enhanced chemiluminescence system (Amersham Biosciences Inc., Piscataway, NJ, USA) followed by exposure to X-ray film (Fuji Photo Film Co. Ltd., Tokyo, Japan), as previously described [25 (link)]. Primary antibodies against iNOS, total p38, phosphorylated JNK, phosphorylated ERK1/2, total JNK, total ERK1/2, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phosphorylated IκB-α, phosphorylated IKK-α/β, and phosphorylated p38 were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

RAW 264.7 macrophages were plated in 6-well plates (4 × 10⁵/well) and cultured in 2 mL of DMEM for 4 h. The cultures were washed to remove non-adherent cells and then incubated with 2 mL of complete DMEM for 20 h. The culture medium was replaced with DMEM for 30 min to allow the cells to adjust. (i) To induce an inflammation model, 1 μg/mL LPS (Sigma) was added. After 24 h of stimulation with LPS, the cells were treated with the indicated concentrations of L-4F (0, 0.1, or 0.25 μg/mL) for 12 h, and the levels of pp38, pJNK and pERK were analyzed. (ii) Cells were stimulated by LPS and treated with the indicated concentrations of L-4F (0, 0.1, or 0.25 μg/mL) for 1 h, and the levels of pSTAT3 were analyzed.
Western blot procedure: Briefly, cells were lysed in radio-immunoprecipitation assay buffer containing the phosphatase and protease inhibitors phenylmethanesulfonyl fluoride and aprotinin (Sigma), and protein was collected. Then, the protein was separated by SDS-PAGE, transferred to PVDF membranes (Roche) and probed with the indicated primary antibodies (phosphorylated STAT3, phosphorylated p38, phosphorylated JNK and phosphorylated ERK; Cell Signaling Technology). The antibody-antigen complexes were detected using a Chemiluminescent HRP substrate kit (Millipore) according to the manufacturer’s protocols.

Antibodies against CXCR7 (ABcam; ab38089), GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA; sc-25778), E-cadherin (Cell Signaling Technology, Danvers, MA, USA; 3195), Ep-CAM (Santa Cruz Biotechnology; sc-25308), N-cadherin (Cell Signaling Technology; 13116), α-smooth muscle actin (Sigma Aldrich, St. Louis, MO, USA; A5228), Slug (Cell Signaling Technology; 9585), Twist (Santa Cruz Biotechnology; sc-81417), Vimentin (Cell Signaling Technology; 3932), phosphorylated-AKT at Ser⁴⁷³ (Cell Signaling Technology; 9271), AKT (Cell Signaling Technology; 9272), phosphorylated-ERK1/2 (Cell Signaling Technology; 9101), ERK1/2 (Cell Signaling Technology; 9102), phosphorylated-JNK (Cell Signaling Technology; 4668), JNK (Cell Signaling Technology; 9251), phosphorylated-p38 (Cell Signaling Technology; 9211), p38 (Cell Signaling Technology; 9212), TGF-β1 (Cell Signaling Technology; 3711), phosphorylated-Smad2/3 (Cell Signaling Technology; 8828), Smad2/3 (Cell Signaling Technology; 8685), MMP2 (ABcam; ab37150), and MMP9 (ABcam; ab38898) were used in Western blotting and immunofluorescence. Small interfering (si) RNAs for controls, CXCR7, and Smad2/3 were purchased from Santa Cruz Biotechnology and Thermo Fisher Scientific (St. Louis, MO, USA). For inhibition of protein kinases, LY294002 and wortmannin were purchased from Sigma Aldrich.

H9‐CMs were grown in 6‐well plates to 80% confluence, detached with TrypLE (Gibco) and then pelleted at 12 000 rpm for 3‐5 minutes at 4°C. After washing with DPBS (Sangon Biotech), the pellets were re‐suspended in 50‐100 μL lysis buffer. Lysates were placed on ice for 30 minutes, and then, the supernatants were collected after centrifuging at 12 000 rpm for 5 minutes. Protein concentration was measured using a BCA kit (Pierce). Western blot was performed using standard protocol with the following antibodies: caspase 3 (Cell Signaling Technology), phosphorylated P38 (Cell Signaling Technology), total P38 (Cell Signaling Technology), total c‐Myc (Cell Signaling Technology), phosphorylated Akt (Cell Signaling Technology) and total Akt (Cell Signaling Technology).

LQ and LQG were purchased from Wako Pure Chemical Industries (Osaka, Japan) and Tokiwa Phytochemical (Tokyo, Japan), respectively. α-Melanocyte-stimulating hormone (α-MSH) was purchased from Sigma Chemical (St. Louis, MO, USA). Antibodies against tyrosinase, TRP-1, TRP-2, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against MITF, phosphorylated CREB, CREB, phosphorylated Akt, Akt, phosphorylated ERK, ERK, phosphorylated p38, and p38 were obtained from Cell Signaling Technology (Beverly, MA, USA). Fetal bovine serum (FBS) was supplied by GIBCO (Gaithersburg, MD, USA). H-89 was purchased from AdipoGen (San Diego, CA, USA). All other chemicals were obtained from Wako Pure Chemical Industries.

Formalin-fixed tissues were decalcified by the 10% nitric acid solution. After deminerazation, the samples were embedded in paraffin, and cut into 4-μm-thicknesses. The prepared slides were dehydrated in xylene and graded ethanol, immersed in citrate buffer and boiled for 10 min using an electronic rage. Immunostaining was performed following the ABC kit manual. After blocking, the slides were incubated with the following primary antibodies at 4 °C overnight: phosphorylated ERK1/2 (1:400; #4370, Cell Signaling Technology, Inc. USA), phosphorylated p38 (1:1600; #4511, Cell Signaling Technology, Inc. USA) and phosphorylated JNK (1:100; #9251, Cell Signaling Technology, Inc. USA). Subsequently, the slides were incubated with a secondary antibody at room temperature for 1 h. All slides were developed using the ImmPACT™ NonaRED™ peroxidase substrate (Sk-4805; Vector Laboratories Inc., USA), mounted and observed under a microscope. To quantify the IHC stain, p-ERK, p-p38 and p-JNK contents were calculated by applying the selected threshold analysis in the positive stain at spine dorsal horns via open-source ImageJ. The positive pixel areas were divided by spine dorsal horn available for the positive stain ratio (positive stain ratio = IHC positive stained area/ the spine dorsal horn area × 100%).

MH-S and PEC were stimulated with HDM and poly(I:C) and CAM or EM900 was added. After 60 min of stimulation, samples were washed with cold PBS buffer and lysed in lysis buffer containing 25% LDS sample buffer (NP0007; Invitrogen) and 5% DTT. Samples were boiled for 5 min, then loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Chicago, IL). Membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich Corp., St. Louis, IL). Membranes were incubated with the specific primary antibodies. After washing with Tris-buffered saline containing 0.1% Tween-20 (TBS-T), membranes were incubated with the secondary antibodies. After washing with TBS-T, membranes were incubated with ImmunoStar® LD containing luminescence solution or ImmunoStar® Zeta containing chemiluminescence solution (Wako). Films were scanned and protein bands were quantified using the C-DiGit® Blot Scanner (LI-COR). Antibodies of nuclear factor-κB (NF-κB) p65, phosphorylated NF-κB p65, p38, and phosphorylated p38 were purchased from Cell Signaling Technology (Danvers, MA) [18 (link)].