Nf kb

The following antibodies were used in Western blotting and immunofluorescence:
phospho-GSK3α/β (CST 8566), phospho-β-Catenin (Ser33/37/Thr41) (CST9561), non-phospho (Active) β-Catenin (Ser33/37/Thr41) (CST 8814), total β-Catenin (CST 9587), phospho-cyclin D1 (CST 3300), PPARγ (CST 2443), p27 (CST 2552), Ki-67 (CST 9129), phospho-c-Jun (CST 9164), c-Myc (CST 13987), Cytochrome c (CST 4280),GFAP (CST 3670), vimentin (CST 5741), NF-kB (CST 8242), c-Fos (CST 2250) from Cell Signaling Technology (Danvers, MA, US); Lamin B1 (10H34L18), Cyclin D1 (MA5-14512), Bcl-2 (13-8800), phospho-CREB (MA1-083), Ki-67 (MA5-14520) from Invitrogen, Thermo Fisher Scientific, and Phospho-JNK (sc-6254) from Santa Cruz Biotechnology (Dallas, TX, USA).

For Western blot analysis, standard SDS-PAGE blotting methods were used. Primary antibodies used in Western blot are as follows: GAPDH, β-tubulin, TLR4, and MYD88 (Beyotime, Beijing, China); ATGL and PPAR-α (Santa Cruz Biotechnology, Inc., Dallas, United States); and CPT-1, SREBP-1c, FASN, ACC, SCD1, and NF-kB (Cell Signaling Technology, Danvers, MA, United States). Chemiluminescence was visualized using an imaging system (330037, Clinx Science Instruments Co. Ltd., Shanghai, China).

α‐Minimum essential medium (α‐MEM) and foetal bovine serum (FBS) were obtained from Gibco; Thermo Fisher Scientific, Inc. Soluble recombinant mouse macrophage‐colony stimulating factor (M‐CSF) and recombinant mouse RANKL were obtained from R&D Systems, Inc. Tartrate‐resistant acid phosphatase (TRAP) was obtained from Sigma‐Aldrich and Merck KGaA. High purity Ti particles (average diameter. 1‐5 µm) were obtained from Johnson Matthey (cat. no., 00681). The antibodies (GAPDH, NFkB, C‐FOS, NFATc1, p‐p38,) were purchased from Cell Signaling Technology, Inc. Enzyme‐linked immunosorbent assay (ELISA) kits (IL‐6, IL‐1β, TNF‐α, IL‐10, Arg‐1, iNOS) were purchased from R&D Systems, Inc Flow cytometry anti‐mouse CD16/32‐PE (cat. no.553145) and anti‐mouse CD206‐Alexa 647 (cat. no.565250) were purchased from BioLegend Inc The p38α/β MAPK inhibitor (SB202190) was purchased from Selleck.

Diaphragm muscles from at least 5 animals/strain (wild type, mdx, and moAb-Il6r-treated and untreated mdx mice) were homogenized in modified lysis buffer (Tris–HCl, pH 7.5/20 mM, EDTA/2 mM, EGTA/2 mM, sucrose/250 mM, DTT/5 mM, Triton-X/0.1%, PMSF/1 mM, NaF/10 mM, SOV₄/0.2 mM, cocktail protease inhibitors/1 × (Sigma) and processed for western blot analysis. Filters were blotted with antibodies against: pStat3 (Tyr705) (cat #9145, Cell Signaling), Stat3 (cat # 9132, Cell Signaling); NFkBp65 (ser536) (cat # 3033, Cell Signaling); and NFkB (cat #4764, Cell Signaling). Signals were captured by ChemiDoc-It® Imaging System (UVP, LLC) and densitometric analysis was performed with VisionWorks®LS Image Acquisition and Analysis Software (UVP, LLC). ELISA assay was performed using either a human (to detect IL6; cat # HS600B) or mouse (to detect Il6, cat # M6000B, and Tnfα, cat # MTA00B) Quantikine® Colorimetric Sandwich ELISAs (R&D Systems), according to manufacturer's protocol. Il2 expression was evaluated on the diaphragm of at least 3 animals/strain and with 2 biological replicates, using a RayBio® Mouse Antibody Array-G series (RayBiotech, Inc.), according to manufacturer's protocol. The intensities of signals were quantified with RayBio® Antibody Array Analysis Tool software.

BV2 cells were seeded on 8-well chamber slides (#80841, ibidi) at a density of 2 × 10⁴ per well. The next day, cells were treated as indicated. Then immunofluorescence assays were all performed according to our standard protocol as described before^{47 (link)}. Primary antibodies used were Nrf-2 (1:250; Cell Signaling), Nf-kB (1:250; Cell Signaling), phosphorylated-P62 (1:500; Cell Signaling), and DAPI (1 μg/ml, Thermo Fisher). Images were collected with a Nikon A1+/A1R+ confocal microscope.