Fusion solo s system

NAc and Striatum were dissected from mice infected with shShank3 or scrShank3 virus. Samples were homogenized at 4 °C with ice-cold lysis buffer (Tris-HCl 50 mM, NaCl 150 mM, Triton 1%) with protease inhibitors (Roche cOmplete™ Protease Inhibitor Cocktail), phosphatase inhibitors (PhosSTOPTM, Roche Diagnostics GmbH) using sequential needle syringe 20G, 26G, and 30G. Protein concentration was calculated using BCA protein assay kit (Thermo Scientific) and samples were separated by electrophoresis onto NuPAGE Bis-Tris 4−12% gel (#NP0322PK2, Invitrogen) and transferred to a nitrocellulose membrane. After 45 min of blocking with TBST-BSA 5%, membranes were probed overnight with primary antibody (anti-Shank3 Neuromab clone N367/62 1:1000, anti-GFP Millipore #MAB3580 1:5000, anti-Tubulin Sigma-Aldrich #T5168 1:10000) diluted in TBST-BSA 1%, followed by incubation with appropriate horseradish peroxidase-conjugated secondary Ab (#1706516 dilution 1:10000 in TBST-BSA 1%). Blots were developed using WesternBright ECL (Advansta) and the chemiluminescence signal was visualized using Fusion solo S system (VILBER). Blots were quantified using ImageJ software.

Cells grown in 6-well plates were washed twice with Phosphate Buffered Saline (PBS) and solubilized with 2% CHAPS buffer (25 mM HEPES-KOH pH 7.5, 300 mM NaCl, 2% [w/v] CHAPS, cOmplete) on ice for 10 min. To detect phosphorylated proteins, PhosSTOP was added to the 2% CHAPS buffer. After centrifugation at 12,000 × g for 2 min at 4 °C, the supernatants were collected, and protein concentrations were determined on a DS-11+ spectrophotometer (DeNovix). SDS-PAGE sample buffer with DTT was added to the supernatants, which were then incubated at 42 °C for 5 min. To detect hAG constructs, the samples were boiled at 95 °C for 5 min. Total cell lysates were loaded on NuPAGE 4–12% Bis-Tris gels and electrophoresed using MES running buffer. Proteins were transferred to PVDF membranes that were blocked with 2% (w/v) skim-milk/TBS-T and then incubated with primary and HRP-conjugated secondary antibodies. Proteins were detected using a Western Lighting Plus-ECL Kit on an ImageQuant LAS4000 (GE Healthcare) or a FUSION SOLO S system (VILBER). ImageJ was used to quantify protein bands. Since the same amounts of total cell lysates were loaded for immunoblotting, the phosphorylated forms pTBK1(pS172), pOPTN(pS177), and p-p62(pS403) were quantified against the total protein.

For Western blot analysis, protein extracts from L2 MVECs stimulated with IFN-γ, PbNK65 extract, IFN-γ and PbNK65 extract in the presence or absence of dexamethasone were separated on SDS PAGE gels and blotted onto a PVDF membrane. Blocking was performed with BSA (Carl Roth Gmbh, Belgium) or non-fat dry milk (Bio Rad, USA). The following specific primary Abs were used: JNK, pJNK, p38, p-p38 (1:2,000, Cell Signaling Technology, The Netherlands), p-GR S211 (1:1,000, Cell Signaling Technology), p-GR S226 (1:1,000, Abcam), GR H300 (1:1,000, Santa Cruz, Germany), p-STAT1 Tyr701 (1:1,500, Cell Signaling Technology), and STAT1 (1:5,000, Cell Signaling Technology). Fusion solo S system (Vilber, France) was used to take chemiluminescence Western blot images. Quantification of Western blot images was performed by densitometry (ImageJ software was used).

Cells were homogenized with lysis buffer (50 mM Tri-Cl (pH 6.8), 10% glycerol, 2% sodium dodecyl sulfate, 1 mM sodium orthovanadate (Na₃VO₄), 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 1 mM 1,4-dithiothreitol). The cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA, #IPVH00010). The membranes were blocked with 5% skim milk solution and incubated with primary antibodies at 4 °C for 12–16 h, then with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature for 2 h. Protein signals were developed with enhanced chemiluminescence (Bio-Rad Laboratories, Hercules, CA, USA, #1705061b) according to the manufacturer’s instructions, and images were observed using a Fusion Solo S system (Vilber Lourmat, Collégien, France).

Western blot was performed as described with slight modifications41 (link). Briefly, cells were lysed by sonication in RIPA buffer (25 mM HEPES, pH 7.8, 0.5 M NaCl, 5 mM EDTA, 1.5% Triton X-100, 1.0% sodium deoxycholate and 0.1% SDS), supplemented with a protease inhibitor cocktail (Roche). After protein concentration measurement, samples were subjected to SDS-PAGE, transferred onto a PVDF membrane (Millipore), and immunoblotted with anti-cyclin D1 (#2922, Cell Signaling Technology), anti-CDK4 (sc-601, Santa Cruz Biotechnology), anti-p27^Kip1 (610242, BD Biosciences), anti-α-tubulin (T9026, Sigma-Aldrich), anti-HIF-1α (610958, BD Biosciences), anti-ferritin light chain (sc-74513, Santa Cruz Biotechnology), or anti-ferritin heavy chain (sc-376594, Santa Cruz Biotechnology). Band intensities were quantified using Fusion Solo S System (Vilber-Lourmat).

Tissue lysates were prepared by homogenization in RIPA buffer [25 mm Tris–HCl (pH 7.6), 150 mm NaCl, 1% NP40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, containing protease inhibitors (cOmplete Mini Protease Inhibitor Cocktail, Sigma Aldrich)], followed by centrifugation at 15,000 rpm for 10 min. Western blotting was performed as described previously (Yamada et al., 2020 (link)). For pS6 and pS6K detection, the membranes were blocked using 1% bovine serum albumin (BSA) fraction V (Fujifilm Wako) in 0.1% Tween 20 in Tris-buffered saline (TBST), followed by incubation with a primary antibody diluted by 1% BSA in TBST. After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Cytiva or Jackson ImmunoResearch). The signal was detected using Immobilon Western chemiluminescent HRP substrate (Merck Millipore). Blot images were captured using the Fusion Solo S system (Vilber Lourmat) and quantified using the Quantification Software Module (Vilber Lourmat).