Ecl substrate

BCA Protein Assay Kit was used to measure the total protein concentration in cells and tissue lysed in RIPA buffer. Then, 20 μg of proteins were separated using SDS‐PAGE at 80 V for 1 h, and the separated proteins were transferred to PVDF. After blocking with 5% skim milk (2 h, 25°C), primary antibodies were incubated overnight at 4°C. The antibodies used were: GAPDH (Abcam), α‐SMA (Abcam), FAP (Abcam), CD63 (Abcam), D81 (Abcam), calnexin (Abcam), IGF1R (CST), AKT (CST), p‐AKT (CST), PI3K (Wanleibio). The diluent used was a special western blotting antibody dilution buffer. The PVDF membranes were then washed and incubated with a secondary antibody conjugated with HRP (Cwbio) (1 h, 25°C). ECL substrate (Invitrogen) was used to obtain protein bands.

Hippocampus and cortex lysates were normalized to 30 μg of protein and separated by SDS-PAGE using NuPage 4 to 12% Bis-Tris gel (Cat#NP0321BOX, ThermoFischer). Protein transfer to polyvinylidene difluoride membranes (Cat#1704156, Bio-Rad) was performed by using the Trans-Blot Transfer system (Cat#1704150, Bio-Rad). Polyvinylidene difluoride membranes were blocked with 3% bovine serum albumin in TBS containing 0.1% Tween-20 (Cat#37520, Cat#28360, ThermoFischer) for 1 h. The membranes were subsequently incubated for 90 min, with the respective primary antibodies: glial fibrillary acidic protein (1:7500 – Cat#16825-1-AP, Proteintech) and GAPDH (1:5000 - Cat#PA1-987, ThermoFischer). The membranes were then incubated for 30 min in horseradish peroxidase–conjugated secondary antibody (1:4000 - Cat#A27036, ThermoFischer). Membranes were developed for 1 min, using ECL Substrate (Cat#WP20005, Invitrogen), and chemiluminescence was captured and quantified by iBright CL1500 Imaging System (Cat#A44240, Invitrogen). Relative protein levels were normalized against the expression of GAPDH, which served as a loading control.

Total cell extracts were obtained using 1X RIPA lysis buffer (Cell Signaling, MA, USA) containing 1X proteinase-inhibitor cocktail (Calbiochem, CA, USA), 1 mM PMSF (Sigma-Aldrich), and 1X phosphatase-inhibitor (Cell Signaling, MA, USA). Cell lysates were fractionated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes (GE Healthcare Life Sciences), and incubated with specific primary antibodies. Immunoblotting was performed with the following antibodies: B7–H3 (R&D System); c-Fos (Abcam, MA, USA); NFATc1 (BD Biosciences, CA, USA); c-Fms, IκBα, p-p38 MAPK, p-p44/42 MAPK, p44/42 MAPK, IDO, p-STAT1, STAT1, AhR, β-actin, and GAPDH (Cell Signaling, MA, USA); RANK, p38α, and iNOS (Santa Cruz, CA, USA). Secondary antibodies (Jackson ImmunoResearch) were incubated before adding the ECL substrate (Invitrogen). For quantitative analysis of protein expression, the optical densities of the blot bands were measured using the Image J software.

Following 72 h treatment, protein samples extracted from MCF7 cells were used for immunoblot assay. Cells were washed twice with ice-cold PBS, and lysed in Cell Lysis Buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerolphosphate, 1 mM Na3PO4, 1 mg/mL Leupeptin, 1 mM PMSF). Cell lysates were centrifuged then normalized by the Lowry assay; proteins in lysates were separated by sodium dodecyl sulfate (SDS)- polyacrylamaide gel electrophoresis (PAGE). Proteins were transferred to nitrocellulose membranes and membranes were blocked with TBST containing 5% non-fat milk. Following overnight incubation of membranes with primary antibodies at 4 °C, membranes were washed with TBST containing 0.1% Tween 20, and incubated with goat antimouse peroxidase- conjugated secondary antibodies for 60 min at room temperature. Blots were probed with an enhanced chemiluminescence (ECL) substrate (Pierce, Thermo scientific) and exposed to Hyperfilm ECL to visualize the immunoreactive bands.

Parotid glands from wild type and Prkcz^−/− mice were lysed in RIPA buffer with 5 mM sodium orthovanadate (Fisher Scientific, Waltham, MA), protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) and 100 mM PMSF (Pierce/Thermo Scientific, Rockford, IL); then, samples were sonicated for 2 minutes and boiled for 10 min until homogeneous. 100 ug of protein from supernatant were loaded in 12% polyacrylamide gels. Transference was performed for 1 hour at 100 V using 0.45 μm Immobilon-P membranes (Millipore, Billerica, MA). After blocking in TBST buffer containing 2% BSA, membranes were incubated at 4 °C overnight in primary antibody at 1:1000 dilution. For detection, HRP conjugated secondary antibodies were applied to the membranes for 1 hour at room temperature and ECL substrate (Pierce/Thermo Scientific) was used following instructions by the manufacturer. The following antibodies were used: anti-total aPKCζ (Cell signaling, #9368), anti-PKCζ (phospho T560) antibody [EP2037AY] (Abcam, ab62372), Anti-Beta-tubulin (Thermo Scientific, #rb-9249-p), anti-Yap (D8H1X) XP Rabbit mAb (Cell Signaling, # 14074), anti-Rabbit-HRP (1:2000, Cell signaling, #7074 S), and Goat-anti-rabbit (1:10000, BioRad, #972-4446).

Tissues samples and cultured neurons were digested in a RIPA buffer with “complete protease and phosphatase inhibitory cocktail” (Roche) and centrifuged (10,000 rpm, 20′). The supernatants were collected, and the amount of total protein was determined by Quick Start Bradford Dye Reagent. Each sample (40 μg) was separated by SDS-PAGE in precast 4–12% Bis-Tris Plus gels (Bolt, Invitrogen), transferred to nitrocellulose membranes (0.45 μM, GE Healthcare), and incubated for 1 h at room temperature with 10% non-fat dry milk in TBS-T (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween-20). The overnight incubation with primary antibody (4 °C) was followed by incubation with the appropriate HRP-conjugated secondary antibody (1:2000, Pierce, 1 h, RT) and the ECL substrate (32106; Pierce- Thermo Scientific). The films were digitized using a professional scanner (HP Scanjet 4050) and quantified by gel densitometry using the ImageJ software (NIH). Measurements were standardized between the experimental groups using the same calibration system and a fixed threshold over the background. Data are expressed as percentage optical density relative to control group, and presented as means ± SEM.