Rabbit anti phospho erk1 2

PDLSCs were washed with ice‐cold PBS and lysed using a RIPA lysis buffer containing 1% PMSF (Solarbio) and 1% phosphatase inhibitor (Boster, Wuhan, China) and then were centrifuged at 12,000 g for 10 min. Protein concentration was measured by a BCA Protein Assay Kit. Proteins were added with SDS‐PAGE loading buffer and then denaturated at 100°C for 5 min. 20 μg/lane proteins were loaded to SDS‐PAGE gels and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with non‐fat dry milk for 1 hr, blotted primary antibodies overnight at 4°C and subsequently incubated with horseradish peroxidase‐labelled secondary antibodies (1:2000; Beyotime, Shanghai, China) for 1 hr at room temperature. The membrane was washed three times in tris‐buffered saline with 1‰ Tween20 (TBST). The immunoreactive bands were visualized using enhanced chemiluminescence reagents (Millipore). The level of each protein was normalized to GAPDH before statistical analysis. Image J 1.44 software (NIH, Bethesda, Maryland, USA) was used to quantify the protein expression. The primary antibodies used were as follows: rabbit anti‐phospho‐ERK1/2 (1:1000; Cell Signaling Technology, Danvers MA, USA), rabbit anti‐ERK1/2 (1:2000; Cell Signaling Technology), rabbit anti‐phospho‐p38 (1:1000; Cell Signaling Technology) and GAPDH (1:10,000; Proteintech, Chicago, IN, USA).

Proteins were separated and detected as previously described [28 (link)]. In brief, SDS-PAGE gel (Mini-Protean TGX Precast Gel 12%, 456–1045, Bio-Rad) was used to separate proteins and then transferred to PVDF membranes (10600023, Amersham Hybond, Pittsburgh, PA, USA). Blocking of membranes was achieved by using 5% dry milk dissolved in Tris Buffer Saline with 1% Tween 20 (TBST) and the following antibodies were incubated overnight at 4 °C: rabbit anti-BCL-2 (CST4223, Cell Signaling), rabbit anti-BCL-xL (CST2764, Cell Signaling), rabbit anti-MCL-1 (CST5453, Cell Signaling), rabbit anti-NOXA (CST14766, Cell Signaling), rabbit anti-BIM (CST2933, Cell Signaling), rabbit anti-phospho-ERK1/2 (CST4376, Cell Signaling), rabbit anti-Actin (CST4970, Cell Signaling). Anti-rabbit IgG HRP-linked secondary antibody (CST7074, Cell Signaling) was used and immunoblots were developed using Clarity ECL Western substrate (1705060, Bio-Rad). When required, immunoblots were stripped in 0.1 M glycine pH 2,5, 2% SDS for 40 min and washed in TBS. The visualization of the bands was done using the LAS4000 imager (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ImageJ was then used to quantify the integrated optical density of bands.

HT22 cells were fixed with 4% paraformaldehyde (PFA) for 15 min, blocked with 10% donkey serum for 1 h at room temperature, and incubated overnight at 4°C with the following primary antibodies: rabbit anti-PSD95 (cat#: ab18258, Abcam, USA), rabbit anti-phospho-ERK1/2 (cat#: 9101, cell signaling, USA), and rabbit anti-phospho-eIF4E (cat#: ab76256, Abcam, USA). The next day, the cells were incubated with donkey anti-rabbit fluorescent secondary antibody (cat#: A21207, Invitrogen, USA) for 2 h at room temperature in the dark. They were then counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (cat#: C0065, Solarbio, China) for 10 min and sealed with anti-fluorescence quenching sealing tablets (cat#: S2100, Solarbio, China). Images were taken using a laser confocal microscope (Olympus, Japan), and the average optical density was analyzed using Fiji software (National Institutes of Health, USA). Intra and inter-assay coefficients of variation were 2.44%-4.95% and 4.07%-9.81% respectively.

Cannabinoids and cannabinoid receptor inverse agonists were purchased from Tocris (Bristol, UK). Murine CCL5 was purchased from Peprotech (London, UK). Murine chemerin and murine CCL2 were obtained from R & D systems (Abingdon, UK). Pertussis toxin (PTX) was purchased from Merck Millipore (Feltham, UK). Bio-gel polyacrylamide beads (P-100 fine 45-90 μm) were purchased from Biorad (Hertfordshire, UK). cOmplete, EDTA free protease inhibitor cocktail tablets were purchased from Roche (Burgess Hill, UK). Rabbit anti-phospho-ERK1/2 and total ERK1/2 were purchased from Cell Signalling Technologies (Danvers, MA, USA). HRP-conjugated Goat anti-rabbit secondary was purchased from Biorad. CIM-16 plates were purchased from Cambridge bioscience (Cambridge, UK). All cell culture media were obtained from PAA systems (Yeovil, UK). Phosphatase inhibitor cocktail 2 and all other laboratory chemicals were purchased from Sigma Aldrich (Dorset, UK). DIAS2 (3-((2’-cyanobenzyl)thio)-5H-[1,2,4]triazino[5,6b]indole) was synthesized by Dr. Angela Russell (University of Oxford, UK)

PBMCs from blood of healthy donors were seeded into 6-well plates at a density of 1 × 10⁶ cells/mL, pretreated with 2 μM JT11 for 1 h, in presence or not of CB2R antagonist SR144528 1 μM, and stimulated with 100 ng/mL LPS (Sigma-Aldrich) for 1 h. Subsequently, cells were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 0.5% Triton X-100, 0.25% Nadeoxycholate, 0.1% SDS, 150 mM NaCl, 1mM EDTA and 5mM MgCl2, including proteases and phosphatases inhibitors). The total protein concentration in each sample was determined by Bradford assay. Protein lysates were analyzed in Western blot as previously described using rabbit anti-phospho-ERK1/2 (Cell Signaling Technology) and rabbit anti-phospho-NF-κB-p65 (Cell Signaling Technology). Immunoreactivity was detected using the ECL Western blotting detection system. As a control for loading of preparation, membranes were stripped and reprobed with rabbit anti-total ERK1/2 (Cell Signaling Technology) or mouse anti-β-tubulin mAbs (Sigma-Aldrich). In parallel experiments membranes were stripped and reprobed with rabbit anti-NF-κB-p65 or with anti-β-actin mAb (Sigma-Aldrich). Densitometric analysis was performed on Mac OS X (Apple Computer International), using NIH Image 1.62 software. The density of each band (absolute value) in the same gel was analyzed.

Protein extracts (25 μg) were resolved by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted with the following primary antibodies: Rabbit anti-SENP1 1/250 (Sigma-Aldrich Cat# HPA011765, RRID:AB_1079907); Mouse anti-PSD95 1/10000 (Antibodies Incorporated Cat# 75–028, RRID:AB_2292909); Mouse anti-Homer1 1/1000 (Synaptic Systems Cat# 160 003, RRID:AB_2631222); Mouse anti-Synapsin1a/b 1/1000 (Santa Cruz Biotechnology Cat# sc-376623, RRID:AB_11150313); Mouse anti-SOD2 1/2000 (Santa Cruz Biotechnology Cat# sc-137254, RRID:AB_2191808); Mouse anti-NOPP140 1/700 (Santa Cruz Biotechnology Cat# sc-374033, RRID:AB_10917069); Rabbit anti-Coilin 1/500 (Santa Cruz Biotechnology Cat# sc-32860, RRID:AB_2081431); Rabbit anti-GM130 1/500 (BD Biosciences Cat# 610823, RRID:AB_398142); Rabbit anti-GAPDH 1/10000 (Sigma-Aldrich Cat# G9545, RRID:AB_79620); Rabbit Phospho-(Ser) PKC Substrate 1/1000 (Cell Signaling Technology Cat#2261, RRID:AB_330310); Rabbit monoclonal Phospho-PKA Substrate 1/1000 (Cell Signaling Technology Cat#9624, RRID:AB_331817); Rabbit anti-ERK1/2 1/1000 (Cell Signaling Technology Cat# 4695, RRID:AB_390779); Rabbit anti-phosphoERK1/2 1/2000 (Cell Signaling Technology Cat# 4370, RRID:AB_2315112). Standard loading controls were included using ERK1/2, GAPDH, PSD95 labelling or ponceau staining as indicated.