Mouse antibody against β actin

Total proteins were extracted from tissues and cells with RIPA buffer (Beyotime, China). Protein concentrations of whole extracts were measured using a BCA protein assay kit (Beyotime, China). Approximately 40μg protein extract each sample was separated using a SDS–polyacrylamide gel and transferred onto the PVDF membrane (Millipore, USA). The membranes were blocked with 5% skimmed milk and incubated with rabbit anti-AKR1B10 (1:500, self-prepared), anti-ERK1/2 (1:1000, abcam, USA), anti-pERK1/2 (1:500, Cell Signaling, USA), anti-MMP2 (1:500, abcam, USA), anti-Vimentin (1:1000, abcam, USA), and mouse antibody against β-actin (1:1000, Sigma, USA) overnight at 4°C, followed by incubation for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000). After extensive washing in PBST, the expression levels of the proteins were detected by Quantity-one software (Bio-Rad Laboratories, USA) using the ECL-chemiluminescent kit.

The following reagents were purchased from the indicated manufacturers: rabbit antibodies against c-Fos, IκBα, RelA, integrin β3, Sphingosine-1-phosphate (S1P); horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG (Cell Signaling Technology, Beverly, MA); mouse antibody against NFATc1, ephrinB2, cadherin 11 (CDH11, OB-cadherin) (Santa Cruz Biotechnology, Dallas, TX); rabbit antibody against p84, cathepsinK, Osterix/Sp7 (Abcam, Cambridge, UK); mouse antibody against β-actin (Sigma–Aldrich, St. Louis, MO); recombinant human M-CSF, β-glycerophosphate, ascorbic acid, and bortezomib (BTZ) (Cell Signaling Technology); carfilzomib (CFZ) (Chemietek, Indianapolis, IN); MLN2238 (ixazomib) (Karebay Biochem, Monmouth Junction, NJ); recombinant human BMP-2 (R&D Systems, Minneapolis, MN); and human-soluble receptor activator of NF-κB ligand (RANKL) (Oriental Yeast, Shiga, Japan).

Linalool, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) (MTT), MPP⁺, MPTP, Hoechst 33342 and mouse antibody against β-actin were obtained from Sigma-Aldrich (St. Louis, MO, USA). DMEM, FBS, and Alexa Fluor 488 goat anti-rabbit IgG were purchased from Invitrogen (Carlsbad, CA, USA). Mouse antibodies against Bcl-2, goat antibody against HO-1 and all horseradish peroxidase-conjugated secondary antibodies were acquired from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibody against caspase-3 and rabbit antibody against PARP were obtained from Cell Signaling (Danvers, MA, USA). Mouse antibody against gp91^phox was purchased from BD Bioscience (San Jose, CA, USA). Rabbit antibody against α-tubulin, rabbit antibody against TH, enhanced chemiluminescence reagent, and polyvinylidene difluoride (PVDF) membrane were procured from Millipore (Bedford, MA, USA). All materials for SDS-PAGE were acquired from Bio-Rad (Hercules, CA, USA).

BRECs and retinas were homogenized in a modified RIPA buffer (20 mM Tris-HCl [pH 7.4], 2.5 mM EDTA, 50 mM NaF, 10 mM Na₄P₂O₇, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate, and 1 mM phenyl methyl sulfonyl fluoride) as described previously [21 (link)]. Equal amounts of protein samples were separated by 12% or 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and reacted for 24 h with anti-arginase 1 (Goat anti-mouse 1:5000, Abcam, Cambridge, MA, USA), anti-p16(INK4a) (Rabbit polyclonal antibody, 1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-NOX2 (Rabbit polyclonal antibody at 1:3000, EMD Millipore Billerica, MA, USA), followed by horseradish peroxidase-linked secondary antibody (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) to detect immunoreactive proteins. Data were quantified by densitometry using NIH ImageJ and normalized to loading control. Equal loading was verified by stripping the membranes and probing them with a mouse antibody against β-actin (1:5000, Sigma-Aldrich, St. Louis, MO, USA). All antibodies were diluted in 2% BSA.

LMMP were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 100 μM NaVO4, 1 mM NaF) containing a mixture of protease inhibitors (0.5 mM EDTA, 0.1 mM PMSF, 1 μM leupeptin, 150 nM aprotinin) (Brun et al., 2013 (link)). Samples were incubated 30 min at 4°C. Particulate material was removed by centrifugation (15,000 × g, 5 min at 4°C) and protein concentration was determined in the supernatants using the bicinchoninic acid assay (Thermo Scientific, MA, USA). Proteins (40 μg/line) were fractionated through SDS-PAGE gel and immobilized onto PVDF membrane (Bio-Rad Laboratories). Unspecific bindings were blocked for 1 h in 5% non-fat dry milk dissolved in PBS and added with 0.05% Tween20. PVDF membranes were then probed with specific antibodies (Table 1). Immune-complexes were revealed using horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma-Aldrich, Italy; Table 1) and enhanced chemiluminescent substrate (ECL, Millipore, Italy). Images were captured using Hyper Film MP (GE Healthcare, Italy). Antibody against mouse β-actin (Sigma) was used as loading control. Densitometric analysis was performed using the ImageJ software (US National Institutes of Health).

Western blot analysis was used to determine expression of the mTOR family of proteins in the placenta and uterine mesometrial compartment of control and treated animals as previously shown (Arroyo et al. 2009). Cell lysates (50 μg) were separated on 4–12% Bis‐Tris gel SDS‐PAGE and transferred to nitrocellulose membranes. Membranes were incubated with antibodies against phospho mTOR (Ser2448), total mTOR, phospho p70 S6 kinase (SK6) (Thr389), total p70^SK6, phospho 4EBP1 (Thr37/46), and total 4EBP1 (all from Cell Signaling Technology, Danvers, MA, excluding total p70 from Epitomics, Burlingame, CA). Membranes were then incubated with a secondary horseradish peroxidase (HRP)‐conjugated antibody for 1 h at room temperature. The membranes were incubated with ECL substrate, and the emission of light was detected using x‐ray film. To determine loading consistencies, each membrane was stripped and reprobed with an antibody against mouse β actin (Sigma Aldrich, St. Louis, MO). Expression levels of the proteins were quantified by densitometry normalized to β actin expression and changes in expression compared to the untreated controls were reported.