Mouse anti β actin

Cell samples were cultured in T25 flasks for 24 h and then lysed on ice using lysis buffer (0.125 M tris at pH 6.8, 4% sodium dodecyl sulfate, and 20% glycerol) with protease and phosphatase inhibitor cocktail (MilliporeSigma). Total protein was quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) and a plate reader (Tecan Infinite 200 Pro). Samples with equal amounts of total protein were subjected to 4%–12% Tris-Glycine gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Then, the blots were blocked with 5% BSA in tris-buffered saline with Tween 20 and incubated with rabbit anti-HRAS (Thermo Fisher Scientific), mouse anti-HER2 (Thermo Fisher Scientific), rabbit anti-pPKCα (Thr638, Thermo Fisher Scientific), or mouse anti-β-actin (Invitrogen) followed by the corresponding Alexa Fluor 680 or 790 conjugated secondary antibodies. The fluorescent bands were imaged using a fluorescent imager (LI-COR Odyssey FC).

Cell pellets were extracted by the M-PER mammalian protein extraction reagent (Thermo Fisher Scientific, 78501) or Mem-PER Plus membrane protein extraction kit (Thermo Fisher Scientific, 89842) with proteinase inhibitors (Roche, 04693159001), and the protein concentration was quantified by a Bio-Rad protein reader. After denaturing at 95 °C for 5 min, the samples (20 μg) were run on 4–15% gradient SDS-PAGE gel at room temperature, and wet transferred onto nitrocellulose membrane at 4 °C. The membranes were incubated in blocking buffer containing 5% (w/v) non-fat milk for 1 h at room temperature, and subsequently incubated overnight at 4 °C in blocking buffer supplemented with primary antibodies: rabbit anti-GFP (1:1,000 Fisher Scientific, A6455), mouse anti-β-actin (1:2,000 Invitrogen, MA5-15739). Fluorophore-conjugated secondary antibodies IRDye® 680RD goat anti-rabbit IgG (LI-COR, 926–68071) and IRDye® 680RD goat anti-mouse IgG (LI-COR, 926–68070) were used at a concentration of 1:10,000 and an incubation time of 1 h at room temperature, followed by infrared imaging.

Protein (30 μg) from the striatum was loaded onto a 10% resolving gel for electrophoresis. Proteins were transblotted onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore) and then immuno-probed with primary antibodies to rat anti-myelin basic protein (MBP, Abcam, Cambridge, UK, ab7349, 1:1000), rabbit anti-CD86 (Abcam, ab112490, 1:1000), rabbit anti-iNOS (Abcam, ab15323, 1:500), rat anti-Fc RII/III receptor (CD16/32, BD Pharmingen, San Diego, CA, 553142, 1:1000), rabbit anti-C3 (Abcam, ab200999, 1:1000), rabbit anti-ITGAM (Abcam, ab133357, 1:1000), goat anti-Arginase-1 (Santa Cruz Biotechnologies, Dallas, TX, sc-18351, 1:500), mouse anti-C3aR (Santa Cruz Biotechnologies, sc-133172, 1:500), rabbit anti-phospho-STAT3 (Tyr705) (Cell Signaling Technology, Danvers, MA, 9145, 1:1000), rabbit anti-STAT3 (Cell Signaling Technology, 4904, 1:1000), mouse anti-β-actin (Invitrogen, Carlsbad, CA, MA5-15739, 1:1000). The blots were incubated with horseradish peroxidase-conjugated IgG secondary antibody (Hua'an, Hangzhou, China) and then reacted with an enhanced chemiluminescence substrate (Pierce, Rockford, IL). The chemiluminescence results were recorded with an imaging system (Bio-Rad, Hercules, CA).

Western blotting was used to assess protein level quantification of ZFP36 in T cells. After primary expansion and prior to cryopreservation, 8 × 10⁶ T cells were pelleted and lysed in ice cold RIPA buffer (G Biosciences) supplemented with protease inhibitor (Sigma-Aldrich) for 30 min on ice then centrifuged at 14,000 rpm for 15 min at 4 °C. All lysates were prepared at a concentration of 1 × 10⁶ cells per 10 µL volume of lysis buffer. Whole cell lysates were boiled for 10 min and then resolved on a 12% Bis–Tris pre-cast gel (Thermofisher). Proteins were transferred onto a polyvinylidene difluoride membrane (Millipore) and then incubated in blocking buffer (LI-COR) for 30 min at room temperature. Primary antibody incubation using rabbit-anti-ZFP36 (Cell Signaling Technologies) and mouse-anti-β-actin (Invitrogen).

Seven weeks after MenSCs transplantation, rats were euthanized by intraperitoneal injection of 250 mg/kg pentobarbital sodium and brain was quickly detached on ice. RIPA buffer supplemented with protease inhibitor cocktail was used to extract protein from brain tissue. After denature, 20 μg of total protein was fractionated by SDS-PAGE. Nonspecific binding was blocked with 5% w/v BSA (A1933, Sigma) in Tris-buffered saline-Tween containing 0.1% Tween-20 (TBST) for 1 h. Primary antibodies, unless specified, were purchased from Proteintech, including rabbit anti-iNOS (18985-1-AP, 1:500), rabbit anti-TH (PA585167, Invitrogen, 1:1000), mouse anti-Arg1 (66129-1-Ig, 1:5000), and mouse anti-β-actin (66009-1-Ig, 1:10000) as the internal control. Primary antibodies were incubated overnight at 4 °C followed by HRP-conjugated secondary antibodies (A16096, Invitrogen, goat anti-rabbit, 1:10000 or 62-6520, Invitrogen, goat anti-mouse, 1:5000) for 2 h at room temperature. Immunoblotted protein signals were visualized with ECL enhanced chemiluminescent substrate kit (WBKLS0050, Millipore) following manufacturer’s instructions. Densitometry of the western blot protein bands was analyzed using Image J software v5.2.1. β-actin was used as an internal control for western blot analysis.

The following primary antibodies were used for either immunofluorescence or Western blotting: rabbit anti-GLUT1 (1:50, cod. 07-1401; Millipore, Burlington, MA, USA); rabbit anti-mTOR (1:50, cod. 2983; Cell Signaling, Danvers, MA, USA); mouse anti-HK2 (1:100, cod. NBP2-02272; Novus Biologicals, Milan, Italy); mouse anti-LAMP1 (1:1000, cod. 555798; BD Biosciences, Franklin Lakes, NJ, USA); rabbit anti-LC3 (1:1000, cod. L7543; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-N-cadherin (1:50, cod. 610920; BD Biosciences, Franklin Lakes, NJ, USA); mouse anti-E-cadherin (1:50, cod. 610404; BD Biosciences, Franklin Lakes, NJ, USA); rabbit anti-TWIST1 (1:1000, cod. T6451; Sigma Aldrich, St. Louis, MO, USA); mouse anti-β-Tubulin (1:1000, cod. T5201; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-IL-6R (1:1000, cod. AHR0061; Invitrogen, Waltham, MA, USA); mouse anti-β-Actin (1:2000, cod. A5441; Sigma-Aldrich, St. Louis, MO, USA).