Radioimmunoprecipitation assay ripa buffer

Mice were anesthetized in a CO₂ chamber and then transcardially perfused with 20 mL ice-cold phosphate buffered saline (PBS). The right hemisphere was post-fixed by incubation for 24 h in 4% PFA followed by impregnation in 30% sucrose until the brains sunk to the bottom. Fixed brains were then cut into 30 µm coronal sections with a cryostat (Leica, Deer Park, IL, USA). The contralateral hemisphere was dissected to isolate dHc, which was hemi-sected: half of the dHc was homogenized in radioimmunoprecipitation assay buffer (RIPA; Sigma-Aldrich, Burlington, MA, USA, R0278; 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate) containing phosphatase and protease inhibitors (Roche, South San Francisco, CA, USA), was centrifuged for 20 min at 15,000× g, and the supernatant collected. The other half of the brain was used for RNA extraction using the RNeasy Mini Kit (Qiagen, Germantown, MD, USA). A separate cohort of mice was anesthetized and perfused with PBS as described above, and the entire hippocampus was isolated and lysed in 8M urea, 10 mM Tris, 100 mM NaH₂PO4, pH 8.5, supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Asheville, NC, USA), as described in [32 (link)].

Cells were lysed with Radioimmunoprecipitation assay buffer (RIPA) (Sigma-Aldrich) supplemented with protease inhibitors (Roche Complete protease inhibitor cocktail, Roche, Basel, Switzerland). Equal amount of lysates were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, MA, USA). Membranes were blocked with 5% w/v milk (Blocking grade milk, Bio-Rad) for 1 h and incubated overnight at 4^oC with primary antibodies against gelsolin (Abcam, UK), Cu/ZnSOD (Cell Signaling, MA, USA), MnSOD (BD), VDAC (Cell Signaling); GAPDH (BD Biosciences) and β-actin (Sigma-Aldrich). The membranes were washed and incubated with horse radish peroxidase-conjugated secondary antibodies. Signals were visualized using chemiluminescence substrate (Thermo Scientific, MA, USA).

Whole cell protein lysate was extracted using Radio-Immunoprecipitation Assay (RIPA) buffer (Sigma Aldrich, St. Louis, MO, USA) plus protease and phosphatase inhibitor cocktail (Sigma-Aldrich). The SDS-PAGE consisting of 12% Acrylamide/Bis gel was used with equal amounts of protein samples. Membranes were incubated for one overnight with the primary antibodies against SOX2 (α-rabbit; 1:1000; Cell Signaling—D6D9, Danvers, MA, USA) or SOX9 (α-rabbit; 1:1000; Cell Signaling—D8G8H). The secondary antibody horseradish radish peroxidase (HRP; Cell Signaling—7074S) was incubated for 1 h at room temperature. Subsequently, signal was then detected by ImageQuant LAS500 system (GE Healthcare Life Science, Chicago, IL, USA) using LumiGLO^® chemiluminescence solution (Cell Signaling—7003S). Beta-Actin (alpha-rabbit; 1:1000; Cell Signaling—D6A8), and/or GAPDH (α-rabbit; 1:1000; Cell Signaling—D16H11), were used as loading for both quantity and quality control of protein lysates.

Dimethyl sulfoxide (DMSO), α-MSH, NaOH, MTT, arbutin, synthetic melanin, Triton X-100, radioimmunoprecipitation assay (RIPA) buffer, skim milk, Tween 20, L-DOPA, and fucoxanthin analytical standard were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin-ethylenediaminetetraacetic acid, TRIzol solution, and bicinchoninic acid (BCA) protein assay kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Antibodies against CREB, p-CREB, β-actin, and anti-rabbit horseradish peroxidase antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Primers against Tyr, Trp-1, Dct, Mitf, Mc1r, and GAPDH genes were synthesized by Bioneer (Daejeon, Korea). An enhanced chemiluminescence (ECL) kit and polyvinylidene fluoride (PVDF) membrane were obtained from Bio-Rad (Hercules, CA, USA). EtOH and EtOAc from DaeJung Chemicals and Metals Co., Ltd. (Siheung, Korea) were analytical grade.