Re blot plus strong solution

FLSs were lysed with RIPA buffer (Thermo Fisher Scientific) supplemented with the Halt™ phosphatase inhibitor cocktail (Thermo Fisher Scientific) and a protease inhibitor mix (Sigma-Aldrich). The protein lysates were fractioned on polyacrylamide gels, followed by electrotransfer to nitrocellulose membranes, which were blocked with either 5% BSA or 5% nonfat dry milk and then incubated with primary antibodies (Cell Signaling Technology: anti-IκBα, anti-IRF1, anti-BAFF, anti-p-STAT1, and anti-STAT1; Sigma-Aldrich: anti-ACTIN). After an incubation with HRP-conjugated secondary antibodies (Cell Signaling Technology), specific bands were detected with the BIORAD Clarity ECL Western substrate. Reprobing was performed using ReBlot Plus Strong Solution (Merck).

Protein samples were obtained from cell lysates using lysis buffer (#9803; Cell Signaling) containing PhosSTOP (#04-906-845-001; Merck) and Complete Protease Inhibitor Cocktail (#11-697-498-001; Merck). Protein samples were reduced with β-mercaptoethanol or dithiothreitol and heated at 95°C with shaking. Samples were loaded on 10% SDS–Page and run at 120 V. Protein samples were transferred onto polyvinylidene fluoride (PVDF) membranes previously activated with methanol. After blocking for 1 h with 4% BSA (#A7906; Merck), the membrane was incubated overnight with primary antibodies diluted in 4% BSA. GAPDH was used for normalisation. Horseradish peroxidase-conjugated antibodies (Jackson Immuno Research) were used as secondary antibodies with incubation for 1 h. Western Lightning Plus-ECL Enhanced Chemiluminescence (NEL105001EA; Perkin Elmer) was used as substrate to develop the membrane in a colorimetric or fluorescence detection. Image SXM or Fusion FX7Edge software were used to quantify protein expression. For multiple stainings, the membranes were re-blotted after stripping with Re-Blot Plus Strong Solution (#2504; Merck).

Whole lysates were collected by lysing cells in radioimmunoprecipitation assay buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with cOmplete, ethylenediaminetetraacetic acid‐free Protease Inhibitor Cocktail (Sigma‐Aldrich) and PhosSTOP phosphatase inhibitor (Sigma‐Aldrich). Immunoblotting was performed by electrophoresing equal amounts of protein (determined by Bicinchoninic acid assays) through precast BOLT gels (Invitrogen), followed by turbo transfer at 25 V for 9 min onto nitrocellulose membranes (Bio‐Rad, Hercules, CA, USA). Membranes were then blocked in 5% skim milk in Tris‐buffered saline containing 0.05% Tween 20, followed by probing with the indicated antibodies (Supplementary table 4). Proteins were visualized using Clarity ECL (Bio‐Rad) and ChemiDoc. Membranes were either stripped using ReBlot Plus Strong Solution (Merck) at room temperature for 15 min or quenched with hydrogen peroxide 30% (Merck) at 37°C for 20 min, enabling reprobing of blots.

Protein samples were obtained from cell lysates using RIPA buffer (Merck, #R0278) containing PhosSTOP (Merck, #04-906-845-001) and Complete Protease Inhibitor Cocktail (Merck, #11-697-498-001). Protein samples were reduced with dithiothreitol and heated at 95 °C with shaking. Samples were loaded on 10% SDS-Page and run at 110 V. Protein samples were transferred onto PVDF membranes previously activated with methanol. After blocking for 1 h with 4% Bovine Serum Albumin (BSA) (Merck, #A7906), the membrane was incubated overnight with Phospho-Akt(S473) (cell signaling, #4058), Phospho-p42/44 MAPK (Erk1/2; T202/Y204) (cell signaling, #9101) or Phopsho-p38 MAPK (T180/Y182) (cell signaling, #9211) diluted in 4% BSA. Total Akt (cell signaling, #9272), ERK (cell signaling, #9102) and p38 MAPK (cell signaling, #9212) were used for normalisation. Horseradish peroxidase-conjugated antibodies (Jackson Immuno Research) were used as secondary antibodies with incubation for 1 h. SuperSignal™ West Pico PLUS Chemiluminescence Substrate (Thermo Scientific, #34,580) was used for protein detection. Quantifiation of protein was done using Fusion FX7Edge software. For multiple stainings the membranes were re-blotted after stripping with Re-Blot Plus Strong Solution (Merck, #2504).

Proteins were applied to SDS-PAGE and electroblotted on a polyvinylidene difluoride membrane. Immunoblotting analysis was performed using a chemiluminescence detection kit. The relative levels of immunoreactivity were determined by densitometry using the ImageJ software.
The primary antibodies were as follows: vesicular acetylcholine transporter (VAChT) (1:500, Synaptic Systems, #139103), GFAP (1:1000, Dako, #Z0334), and Actin (1:10000, Millipore, #MABT825). The secondary antibodies were as follows: goat anti-mouse IgG (1:3000; Bio-Rad, # 1706516) and goat anti-rabbit IgG (1:3000; Bio-Rad, # 1706515).
Membranes were stripped using Re-Blot Plus Strong Solution (Millipore) for 15 min at room temperature.

Hypothalamic lysate samples containing 40 µg of protein were separated on 4%–20% gradient polyacrylamide TGX gels (BIO-RAD, Hercules, CA, USA; 5671094) via electrophoresis and transferred to 0.45 µm pore size Immobilon-FL polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA; IPFL00010). Membranes were incubated in PBS-based blocking buffer (LI-COR Biosciences, Lincoln, NE, USA; P/N 927) for 1 h and then probed with primary antibodies overnight at 4 °C with shaking, followed by washing with PBS containing 0.01% Tween 20 (PBS-T). Blots were incubated with infrared fluorophore-bound secondary antibodies in the dark, washed again with PBS-T, and analyzed using the Odyssey CLx Imaging System (LI-COR Biosciences). After the phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was measured, membranes were stripped (Re-Blot Plus Strong Solution; Millipore, Burlington MA, USA; 2504) and then probed for STAT3 to generate a pSTAT3/STAT3 ratio.