Anti rabbit igg hrp sc 2357

Cells were harvested in lysis buffer including 100 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 1 mM Na3VO4, 1 mM NaF, and 1 mM PMSF, 1 mM PIC, 1 mM PhiC. Cell lysates were separated by SDS-PAGE and transferred into PVDF membranes. Primary antibodies used were: RARα (sc-515796), RARβ (sc-552), RARγ (sc-7387), Src (sc-5266), LDH (sc-133123), p-Paxillin (sc-365020) and Paxillin (sc-31010) from Santa Cruz Biotechnology (Santa-Cruz, CA, USA); p-FAK (BD-611807) and FAK (BD-610088) from BD Transduction Laboratories; cleaved PARP1 (ab32064) from Abcam (Cambridge, UK), active caspase-3 (bs-0081R) from Bioss. Secondary antibodies (Bioss Antibodies, Woburn, MA, USA) used were: anti-rabbit IgG-HRP (sc-2357), anti-mouse IgG-HRP (sc-358914) and anti-goat IgG-HRP (sc-2354) from Santa Cruz Biotechnology. Primary and secondary antibodies were incubated using the standard techniques. Immunodetection was accomplished using enhanced chemiluminescence and was recorded with a quantitative digital imaging system (Chemidoc XRS with Image Lab, Bio-Rad, Hercules, CA, USA).

The WT BMDMs were harvested and lysed in NP40 Cell Lysis Buffer (FNN0021, Invitrogen). The lysates were centrifuged at 15,300× g for 10 min at 4 °C, and the supernatants were obtained. The protein concentrations of the supernatants were determined by applying the Bradford assay (500-0006, Bio-Rad Laboratories, Hercules, CA, USA). Proteins were electrophoresed on NuPAGE 4−12% Bis-Tris gels (Invitrogen) and transferred to Protran nitrocellulose membranes (10600001, GE Healthcare Life Science, Pittsburgh, PA, USA). The membranes were blocked in 5% (w/v) bovine serum albumin (BSA) (9048-46-8, Santa Cruz Biotechnology) in TBS-T (TBS (170-6435, Bio-Rad Laboratories) and 1% (v/v) Tween-20 (170-6531, Bio-Rad Laboratories)) for 30 min at 25 °C. The membranes were incubated with primary antibody diluted in 1% (w/v) BSA in TBS-T for 16 h at 4 °C and then with the horseradish peroxidase (HRP) conjugated secondary antibody (anti-rabbit IgG-HRP (SC-2357, Santa Cruz Biotechnology) (1:2500) and anti-mouse m-IgGκ BP-HRP (SC-516102, Santa Cruz Biotechnology) (1:2500)) diluted in TBS-T for 30 min at 25 °C. The immunoreactive bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (34078, Thermo Scientific, Waltham, MA, USA).

FNT (formononetin) was purchased from TargetMol (Boston, MA, USA). 4-nitrophenyl N-acetyl-β-D-glucosaminide was purchased from Sigma (St. Louis, MO, USA). Dexamethasone (Dexa), ketotifen fumarate (Keto), Evans blue, and toluidine blue were purchased from Meilun Biotechnology (Dalian, China). Primary rabbit antibodies against IκBα (ab76429, monoclonal), p-IκBα (ab133462, monoclonal), p65 (ab32536, polyclonal), GAPDH (ab181602, monoclonal), and loricrine (ab176322, monoclonal) were purchased from Abcam (Cambridge, MA, USA). Antibodies targeting filaggrin (DF13853, monoclonal) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Anti-p-p65 (sc-135769, monoclonal), anti-rabbit IgG-HRP (sc-2357, monoclonal), and 4% paraformaldehyde solution in PBS were from Santa Cruz Biotechnology (Dallas, TX, USA). 2,4-dinitrochlorobenzene (DNCB) was purchased from Tokyo Chemical Industry (Tokyo, Japan). C48/80 was purchased from Cayman Chemical (Ann Arbor, MI, USA).