Odyssey sa infrared imaging system

Retinal tissue from wt and rd1 mice were homogenized in modified RIPA lysis buffer (50 mM trizma base, 150 mM NaCl, 19 mM Na₄O₇P₂, 1 mM EDTA, 1 vol% Triton-X-100, 1 mM DTT and 0.1 vol% of protease inhibitor cocktail III EDTA-free (EMD Millipore Corp., Billerica, Massachusetts, USA); pH = 7.4) with a Precellys homogenisator (Bertin Technologies, Montigny le Bretonneux, France). For separation of proteins, 25 μg protein per well were loaded onto a 12% SDS-PAGE gradient gel, and run at 120 V. Subsequently, the proteins were transferred to PVDF membranes (Merck Millipore, Tullagreen, Ireland). Roti block buffer (Roth, Karlsruhe, Germany) was applied for 3 h at room temperature. Membranes were incubated in primary antibodies against PAR (1:1000; see above) or actin (1:400; Abcam, Milton, UK; Ab1801) in buffer containing PBST and 5% dried milk (Carl Roth GmbH, Karlsruhe, Germany) overnight at 4 °C. Membranes were washed with PBST and incubated with secondary antibodies labelled with IRDye680 RD (LI-COR Biotechnology GmbH, Bad Homburg, Germany; 926–68070) or IRDye800 CW (LI-COR Biotechnology GmbH, Bad Homburg, Germany; 926–32211) for 1 h at room temperature. LI-COR Odyssey Sa Infrared Imaging System (LI-COR Biotechnology GmbH, Bad Homburg, Germany) was used for detection of fluorescent protein bands, which were quantified using ImageJ (National Institute of Health, Washington, USA).

Granulosa cells were washed with PBS, lysed with RIPA buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% sodium deoxycholate, 5 mM EDTA) supplemented with protease inhibitors cocktail (Roche) and 1 mM PMSF. Equal amounts of total protein were separated by SDS/PAGE gels, transferred to nitrocellulose membrane, and probed with the primary antibodies. The images were captured with the ODYSSEY Sa Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Densitometry was performed using ImageJ software. The protein expression was normalized to that of GAPDH. Blots are representative of three independent experiments. The antibodies used were PRMT5 (Millipore, 07-405), MEP50 (Abcam, ab154190), WT1 (Abcam, ab89901), FOXL2 (Abcam, ab5096), CYP11A1 (Proteintech, 13363-1-AP), StAR (Santa Cruz, sc-25806), SF1 (Proteintech, 18658-1-AP), and FLAG (Sigma, F1804).

The protein expression of synapse related genes was determined by western blot as described in our previous study42 (link). Briefly, the proteins were separated by electrophoresis on an 8% or 6% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene fluoride membranes. The membranes were incubated with anti-human synapsin 1 (1: 6,000, CST), NRXN1 (1: 1,000, Abcam), NLGN3 (1: 1,000, Abcam), SHANK3 (1: 200, Santa Cruz), PSD-95 (1: 2,000, CST), and β-actin (1: 5,000, CST) antibody overnight at 4 °C. Then, the strips were incubated with anti-rabbit or anti-mouse or anti-goat IRDye 800CW IgG (1: 15,000, LI-COR) for 1 h at room temperature. The immunoreactivity signals were directly detected using an Odyssey SA Infrared Imaging System (LI-COR Biosciences, USA). The respective intensities were determined using Quantity One software.

Cells were washed twice in PBS, lysed in 1× Glasgow lysis buffer (GLB; 1% [vol/vol] Triton X-100, 120 mM KCl, 30 mM NaCl, 5 mM MgCl₂, 10% [vol/vol] glycerol, and 10 mM PIPES-NaOH, [pH 7.2], with protease and phosphatase inhibitors) and harvested by centrifugation (2,800 × g, 10 min, and 4°C) before determination and normalization of protein concentration by bicinchoninic acid (BCA) assay (Pierce). Following separation by SDS-PAGE, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 50% (vol/vol) Odyssey blocking (OB) buffer (LI-COR) in Tris-buffered saline (TBS). The membrane was incubated with primary antibodies overnight at 4°C, followed by secondary antibodies for 2 h at room temperature, both prepared in 25% OB buffer. Primary antibodies used were anti-NS5A (sheep, prepared in-house) at 1:4,000 (52 (link)), anti-NAP1L1 (rabbit; Santa Cruz) at 1:350, anti-Bin1 (rabbit; Generon) at 1:300, anti-VAP-A (rabbit; Generon) at 1:1,000, anti-VAP-B (rabbit; Generon) at 1:1,000, and anti-α-actin (mouse; Sigma) at 1:10,000. Secondary antibodies were anti-rabbit, anti-sheep (800 nm), or anti-mouse (700 nm) antibodies, used at 1:10,000 prior to imaging using a LI-COR Odyssey Sa infrared imaging system. Quantification of Western blots was carried out using Image Studio v3.1 (LI-COR) using a background subtraction method.