Phospho p38 mapk antibody

HH cells were cultured in six-well plates in the presence or absence of anti-human CD147 antibody (1 µg/mL; Biolegend) or anti-human CypA antibody (0.7 µg/mL; Abcam) for 1, 3, or 6 h. After collecting proteins from HH cells, equal amounts of proteins were subjected to 4–12% NuPage Bis-Tris Gels (Invitrogen, Waltham, MA, USA) at 200 V for 20 min. The proteins were then transferred onto polyvinylidene fluoride membranes (Invitrogen) and blocked in 2% skim milk powder with 0.05% Tween-20 (Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline. The membranes were probed with Akt Antibody (Cell Signaling Technology, Danvers, MA, USA), Phospho-Akt Antibody (Cell Signaling Technology), ERK1/2 Antibody (Cell Signaling Technology), Phospho-ERK1/2 Antibody (Cell Signaling Technology), p38 MAPK Antibody (Cell Signaling Technology), Phospho-p38 MAPK Antibody (Cell Signaling Technology), SAPK/JNK Antibody (Cell Signaling Technology), Phospho-SAPK/JNK Antibody (Cell Signaling Technology), or β-actin Antibody (Cell Signaling Technology) as primary antibody overnight at 4 °C, followed by incubation in secondary antibody for 30 min at room temperature. Visualization was performed by SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA).

Protein extraction, gel electrophoresis, and Western blotting procedures were carried out as described previously [4 (link)]. Primary antibodies (Cell Signaling Technology, Danvers, MA), along with their concentrations, were phospho-p38 MAPK antibody, #9211 (1:1,000); phospho-p44/42 MAPK mAb, #4370 (1:2,000 or 1:3,000); and phospho-eIF2α mAb, #3398 (1:1,000). The secondary antibody (1:20,000) was peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA).

Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to NC membranes, and blocked with TBST of 5% skim milk for 1 hour. The membranes were washed with TBST and then incubated with the specific primary antibody for 6 hours at 4°C. The membranes were then incubated for 1 hour at room temperature in secondary and the signal was detected by chemiluminescence (Bio rad, USA). The primary (1:1000) and secondary (1:10000) antibodies were purchased from Cell Signaling Technology, USA and listed as follows: GAPDH antibody(#2118), stat3 antibody(#12640), phospho-stat3 antibody(#98543), SAPK/JNK antibody(#9252), phospho-SAPK/JNK antibody(#4668), p65 antibody(#4764), phospho-p65 antibody(#3033), IKKα antibody(#2682), phospho-IKKα/β antibody(#2697), IκBα antibody(#4812), phospho-IκBα antibody(#2859), p38 MAPK antibody(#8690), phospho-p38 MAPK antibody(#4511), Erk1/2 antibody(#4695), phospho-Erk1/2 antibody(#4370) and HRP-linked goat anti-rabbit IgG(#7074).

All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), if not specified. RPMI1640, MEM, fetal bovine serum (FBS), penicillin/streptomycin, and trizol reagent were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibody of Lamin B, I-κB-α rabbit polyclonal antibody, β-actin mouse monoclonal antibody, NF-κB p65 rabbit polyclonal antibody, phospho-ERK antibody, MMP-7 goat polyclonal antibody, p67phox goat polyclonal antibody, NOX1 rabbit polyclonal antibody, NOX2 goat polyclonal antibody, celecoxib were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phospho-p38 MAPK antibody, ERK antibody, p38 MAPK antibody, phospho-AMPK-α, AMPK-α, phospho-JNK, JNK, C-Jun, phospho I-κB, and PD98059 were purchased from Cell Signaling Technology, Inc. (Boston, MA, USA). Matrigel was obtained from BD Biosciences (Bedford, MA, USA). Trypsin/EDTA was purchased from Clonetics, Inc. (Walkersville, MD, USA). SR11302 was purchased from Tocris Bioscience (Tocris House, Bristol, BS110QL, UK). D942 was purchased from Calbiochem (10394, Pacific Center Ct, CA, USA).

The media and other materials required for culturing cells were purchased from Lonza Inc. (Walkersville, MD, USA). Griess reagent and lipopolysaccharide (LPS: E. coli, serotype O111:B4; L2630) were purchased from Sigma–Aldrich (St. Louis, MO, USA). The WST-1 assay kit was obtained from Daeillab Service Co., Korea. Anti-Toll-like receptor 2 antibody (anti-TLR2), anti-complement receptor 3 antibody (anti-CR3), and anti-Toll-like receptor 4 antibody (anti-TLR4), were obtained from Abcam (Cambridge, MA, USA). Phospho-NF-κB antibody, phospho-p38 (MAPK) antibody, phospho-ERK (MAPK) antibody, and phospho-JNK antibody were purchased from Cell Signalling Technology (Danvers, MA, USA). All chemicals and reagents used in this work were of analytical grade.