Goat anti mouse igg h l

RIPA buffer was added to cells for incubation at 4°C for 10 min, then the supernatant(cells lysates) was collected by centrifugation. Cell lysates and isolated sEVs were subjected to 12% SDS-PAGE electrophoresis and western blotting according to standard protocols. After electrophoresis, the proteins are transferred from the gel to a nitrocellulose filter membrane by electrotransfer. The nitrocellulose filter membrane was incubated at 4 °C for 16 h with the indicated primary antibodies against MFG-E8, EGFP, CD9, CD63, luciferase, or αvβ3 (Invitrogen, Cat.No. PA5-82036; Proteintech, Cat.No. 66002–1-Ig; 60232–1-Ig; 67605–1-Ig; 67293–1-Ig; 66952–1-Ig; Abcam, Cat. No. ab190147) and then washed for three times in Tris-buffered saline T (TBS-T), followed by 1 h incubation with Goat Anti-Mouse IgG(H + L) (Proteintech, Cat.No. SA00001-1) at room temperature.

Renal tissues were obtained from patients with LN (IV + V) by biopsy and without renal autoimmune disease by kidney surgery. Tissue immunohistochemistry and PBMC cellular immunofluorescence were performed to detect cGAS and KAT2A proteins. Paraffin-embedded renal tissues were used for immunohistochemistry. 5−10% goat serum was used for blocking, then samples were incubated with primary cGAS antibody overnight (4 °C). After washing for three times, samples were incubated for 30 min with biotin-binding secondary antibody. The liquid was dropped on samples which then were been washed for three times with PBS. HRP-Streptavidin diluted with PBS was added and incubated for 10 min at 37 °C. Samples were washed for three times with PBS, AEC was used for visualization.
PBMCs were fixed and permeabilized in 0.3% Triton for 30 min. Non-specific sites were blocked with goat serum for 60 min at 37 °C. Samples were incubated with primary antibodies (KAT2A, cGAS) overnight at 4 °C. Secondary goat anti-mouse IgG (H + L) (1:200, Proteintech) were incubated with samples for 90 min at 37 °C. DAPI (Wellbio) was used for nucleus staining for 10 min. After washing for three times with PBS, samples were observed under fluorescence microscope (×400). Positive signals were shown as green fluorescence for cGAS, red for KAT2A, blue for DAPI.

D4, D6, and D8 myocytes were stained with MYHC immunofluorescence to observe myotube changes. The cells were fixed with 4% tissue cell fixation solution (Solarbio) for 10 min; permeated with 0.2% TritonX-100 (Sigma, Kawasaki, Japan) for 20 min; incubated with phosphate-buffered saline (PBS, Hyclone) containing 10% donkey serum (Solarbio), 1% bovine serum albumin (Sangon Biotech, Shanghai, China), and 0.3 M glycine (Sigma, Kawasaki, Japan) for 1 h; incubated with MYHC antibody (mouse anti-MYHC, 1:200, Abcam, ab50967, Cambridge, NT, UK) at 4 °C overnight; then washed with PBS 3 times. Before imaging, the cells were incubated at 37 °C for 1 h with Goat Anti-Mouse IgG (H + L) (1:200, SA00013-3, Proteintech Group, Inc., Rosemont, IL, USA), the cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, Gibco, Grand Island, NY, USA) for 15 min, and it was washed 3 times with PBS before being imaged on an Olympus IX71 microscope (OLYMPUS 100×, Dalian, China).

The proteins of suspensions OFs, exosomes, and tissues were obtained and quantified by the BCA method (Beyotime, Shanghai, China). Protein lysates were transferred to the PVDF membrane by 8% SDS-PAGE (Beyotime Biotechnology, China). The PVDF membrane was cleaned with TBST and sealed with 5% skim milk powder for 2 h. The cut film was incubated overnight in the primary antibody dilution solution and 2 h in the secondary antibody dilution solution. Finally, the film was scanned. The main antibodies used included HRP-binding GAPDH monoclonal antibody (ProteinTech, Wuhan, China), AKT polyclonal antibody (ProteinTech, China), NFκB P65 polyclonal antibody (ProteinTech, China), Rabbit Anti-phosphorylated NFκB P65 (Ser276) Antibody (Bioss, Beijing, China), Rabbit Anti-phosphorylated AKT (Ser473) antibody (Bioss, China), IL-1α polyclonal antibody (ProteinTech, China), ICAM1 polyclonal antibody (ProteinTech, China), CD9 monoclonal antibody (ProteinTech, China), TSG101 polyclonal antibody (ProteinTech, China), CD81 monoclonal antibody (ProteinTech, China), Goat Anti-mouse IgG (H+L) (ProteinTech, China), Goat anti-rabbit IgG (H+L) (ProteinTech, China), and glucocorticoid receptor polyclonal antibody (ProteinTech, China).

The proteins were extracted by lysing the kidney tissue with RIPA (Solarbio, China). The concentration of the proteins was measured by a BCA protein assay kit (Solarbio, China). The proteins were mixed with 4×SDS buffer (Solarbio, China), heated for 10 min and then separated on 10% SDS-PAGE gels. Proteins were transferred to PVDF membranes (Millipore, USA), and the membranes were blocked with TBST (containing 5% skim milk) at room temperature for 1 h. Then, the membranes were incubated with rabbit anti-GLUT9 antibody (1:2500, Millipore, USA) and rabbit anti-β-actin antibody (1:5000, Proteintech, USA) at 4°C overnight. The next day, the immunoreactive bands were detected using goat anti-rabbit IgG H&L (1:10000, Abcam, USA) and goat anti-mouse IgG H&L (1:10000, Proteintech, USA) as the secondary antibody at room temperature for 1 h. The protein blots were visualized using ECL immunoblot detection reagent (Millipore, USA). The density of bands was analysed by Image J and normalized to β-actin. Protein expression was quantified as the ratio of the specific band to β-actin.

Phenylephrine (PE) was purchased from Tokyo Chemical Industry (P0398). Hispidulin (SML0582), Dimethyl sulfoxide (DMSO) (D2650), and EX-527 (E7034) were obtained from Sigma-Aldrich (St. Louis, USA). With the purpose of detecting specific proteins by Western blotting, the following primary antibodies, obtained from Cell Signaling Technology (Danvers, MA, USA), were applied: anti-AMPK (Thr172) (#2531), antiphosphorylated AMPK (#2532), anti-Sirt1(D1D7) (#5490) antibody, anti-phosphorylated p38 (Thr180/Tyr182) (#4511), anti-p38 (#8690), anti-phosphorylated Akt (Ser473) (#4060), anti-Akt (#9272), anti-phosphorylated JNK1/2 (Thr183/Tyr185) (#4668), anti-JNK1/2 (#9252), anti-Tom20 (D8T4N) (#42406), anti-OPA1 (D6U6N) (#80471), anti-Mitofusin2 (D1E9) (#11925), and anti-GAPDH (#2118) antibodies. Anti-PGC-1α antibody (66369-lg) and secondary antibodies, including goat anti-rat IgG (H+L), horseradish peroxidase (HRP) conjugate (SA00001-15), goat anti-mouse IgG (H+L), and HRP conjugate (SA00001-1), were obtained from Protein-tech Group (Wuhan, China). The anti-vinculin antibody was purchased from Sigma-Aldrich (V9264).

Antibodies were incubated with Dynabeads protein G (Invitrogen, Carlsbad, US) at 4°C for at least 30 minutes. Cells were lysed in NP-40 buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail (Thermo Fisher, Waltham, US). Cell lysate was incubated with antibody-coated beads at 4 °C overnight. Antibodies used for immunoblotting and co-immunoprecipitation included anti-Vam6 (Thermo Fisher, Waltham, US), anti-VDAC1 (Abcam, Cambridge, UK), anti-Rab7a (NewEast, Kelayres, US), anti-AMPKγ (Invitrogen, Carlsbad, US), anti-mCherry (Invitrogen, Carlsbad, US), anti-β-actin (Proteintech, Chicago, US), and rabbit IgG isotype ctrl (Invitrogen, Carlsbad, US). HRP-conjugated secondary antibodies included mouse-anti-rabbit IgG light chain (CST, Danvers, US), goat-anti-mouse IgG (H+L) (Proteintech, Chicago, US), and goat-anti-rabbit IgG (H+L) (Proteintech, Chicago, US). Antibodies and isotype controls used in co-immunoprecipitation experiments were at 1 μg/mL, and antibodies for immunoblotting were used at 1:1000 dilution unless other described.