Odyssey clx

Approximately 1 × 10⁵ HEK293 cells were seeded in flat-bottom 24-well plates (Thermo Fisher Scientific) and incubated overnight. The following day, the cells were cotransfected with the Cas13 variant-expressing plasmid (pXR series, 150 ng), the gRNA-expressing plasmid (pC016-gPsp-Control, 300 ng), and the luciferase reporter plasmid (psiCHECK2-PTGES3, 5 ng) using Lipofectamine 3000 according to the manufacturer’s instructions. Forty-eight hours posttransfection, the cells were treated with 20 μM OP-puro (Jene Bioscience) for 30 min at 37 °C with 5% CO₂, washed with PBS, lysed in OP-puro lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 1% Triton X-100), and then clarified via centrifugation at 20,000 × g for 10 min at 4 °C. The lysates were incubated with 1 μM IRDye800CW Azide (LI-COR Bioscience) for 30 min at 25 °C using a Click-it cell reaction kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and subsequently subjected to SDS‒PAGE. Images from the gels were acquired and quantified using an ODYSSEY CLx (LI-COR Biosciences). Total protein staining was subsequently performed on the gels using GelCode Blue Safe Protein Stain (Thermo Fisher Scientific). The proteins were quantified using an ODYSSEY CLx (LI-COR Biosciences) and used for the normalization of OP-puro-labeled nascent protein signals.

Expression of pp38 in MSB-1-Δpp38 and HP8-Cas9-Δpp38 cells before and after sodium butyrate (NaB) or 5-Azacytidine (AZA) treatment was determined by western blot analysis using anti-pp38 Mab BD1 as the primary antibody. Mab against MDV Meq (FD7, generated at Pirbright Institute) and α-tubulin (Sigma Aldrich) were used as a control. Briefly, 1 × 10⁶ cells were collected before and after treatment with 0.5 mM NaB (Sigma) or 10 µM AZA for 72 h and boiled with TruPAGE^TM LDS sample buffer (Sigma) for 10 min. The samples were separated on a 4–12% TruPAGE^TM Precast Gel, and the resolved proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. Immunoblots were blocked with 5% skimmed milk, and then incubated with anti-pp38 and anti-Meq antibodies. After probing with primary antibodies, the blots were incubated with secondary antibody IRDye^® 680RD goat anti-mouse IgG (LI-COR, Lincoln, NE, USA) and visualized using Odyssey Clx (LI-COR). For α-tubulin detection, the PVDF membranes were incubated in stripping buffer (100 mM 2-Mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl PH 6.7) for 30 min at 50 °C, and then blocked with 5% skimmed milk after washing in PBS twice for 10 min. The same procedure was followed for detection of α-tubulin and visualized by Odyssey Clx (LI-COR).

Plasma was used to validate chemerin presence in the WT and loss in the KO rats. One hundred micrograms total protein derived from each animal were loaded per lane on standard polyacrylamide gels (15%). Gel proteins were transferred to a PVDF-FL membrane using wet transfer. A LI-COR REVERT® kit (Lincoln, NE, USA) was used to stain total protein and scanned on 700 channel of a LICOR Odyssey CLx (Omaha Nebraska, USA). The membrane was then rehydrated in methanol, rinsed in water, and total protein stain removed with Reversal solution for 5 min, then rinsed twice. All blots were then blocked in 4% chick egg ovalbulmin (catalog #A5378, Sigma, Sigma Chemical Company, St. Louis, MO, USA) for three hours on a rocker at 4 °C. Chemerin antibody (catalog #112520, Abcam, Cambridge, MA USA; RRID:AB_10864055) diluted to 1:1000 in blocking buffer was incubated for 48–72 h with constant rocking at 4 °C. Blots were then rinsed with tris-buffered saline + 0.1% Tween-20 three times for 10 min, followed by incubation with LICOR IRDye secondary antibody (catalog # 926-32210, 800CW goat anti-mouse at 1:1000, Lincoln, NE, USA; RRID:AB_621842) for an hour with rocking. Secondary solution was removed, blot was washed 3 × 10 min with tris-buffered saline + Tween, and imaged on a LICOR Odyssey CLx in the 800 channel.

Total proteins from spinal cord samples and cultured cells were extracted and used for Western blots as described in [23 (link)]. Proteins were detected using the Odyssey CLx Li-Cor technology (Li-Cor, Bad Homburg, Germany). Briefly, primary antibodies were incubated in Li-Cor PBS buffer overnight at 4 °C. After washing, membranes were incubated with secondary fluorescent dye (IRDye 800CW for OSMR protein and IRDye 680LT for β-actin normalization). For Figure 5D–F, signals were obtained with peroxidase-conjugated secondary antibodies (Cell Signalling Technology, Danver, MA, USA) and revealed with Clarity Western ECL kit (BioRad, Marnes-la-Coquette, France) and a ChemiDoc™ XRS Imaging system (BioRad).

LDS preparations were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were incubated overnight at 4 °C in primary antibodies diluted in 3% milk in PBS-Tween. The following day, membranes were washed and incubated at room temperature for 1 h in secondary antibodies diluted in PBS-Tween. Protein bands were detected using Odyssey CLx LI-COR.

A fraction of lentivirus-infected cells was lysed in NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA). The lysates were denatured in 5 × SDS loading buffer by boiling at 95 °C for 10 min and were subjected on a NuPAGE™ 4–12% Bis–Tris Protein Gels (Invitrogen). After transferred to Biotrace NT nitrocellulose Transfer Membrane (PALL, 66,485), the expression of proteins was detected using following antibodies: 1:300 DNMT3A (D23G1) Rabbit mAb (No. 3598; CST) alone was incubated firstly, then 1:300 DNMT3A (D2H4B) Rabbit mAb (No. 32578; CST) and 1:2000 Lamin B1 Mouse mAb (No. 66095–1-Ig; Proteintech) were mixed and incubated together on the next day in the same blot after finishing the secondary antibody incubation and band scanning for DNMT3A(D23G1) mAb. Secondary antibody included Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 (No. A21058; Invitrogen) and Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (No. A32735; Invitrogen). The bands were scanned by LICOR Odyssey CLX.