Nf κb p65 rabbit mab

Dynabeads coupled with Anti-Rabbit secondary antibody (M-280; Thermo Fisher Scientific) were resuspended in 1 ml of Washing Buffer (Ca2 + and Mg2 + free (PBS), supplemented with 0.1% bovine serum albumin (BSA) and 2 mM EDTA, pH 7.4). With the help of a DynaMag^TM−2 Magnet, the Dynabeads were washed. 5% BSA and NF-κB p65 Rabbit mAb (Cell Signalling Technology) were added to the beads and incubated with gentle tilting and rotation at RT for one hour. The unbound NF-κB p65 Rabbit mAb was removed using a DynaMag^TM-2 Magnet. Dynabeads were washed with 5% BSA to ensure all unbound NF-kB p65 Rabbit mAb was removed.
These Dynabeads were then resuspended in the nuclei in 5% BSA and incubated for over 12 hours at 4 °C. The product obtained after the incubation was a tertiary complex comprised of Dynabeads coated with Anti-Rabbit secondary antibody, bound to NF-κB p65 Rabbit mAb, which was further bound to chromatin associated with NF-κB p65. The beads were washed with PBS, to ensure that the chromatin that was not associated with NF-κB p65 was washed off.

For western blotting analysis, the proteins of lung samples and cell lysates lysed by RIPA buffer (Beyotime, Shanghai, China) were separated using SDS-polyacrylamide gel electrophoresis. Subsequently, the proteins resolved in the gel were transferred to the polyvinylidene fluoride membrane (Millipore, MA, USA). Western blots were visualized using the ECL system (Fdbio Science, Hangzhou, China) and Quantity One software (Bio-Rad, USA) was used to analyze the intensity of individual bands. Data were normalized with β-actin as a loading control in each individual sample. The following antibodies were used in western blotting analysis: influenza virus NP rabbit pAb (1:2000), IFITM3 rabbit pAb (1:2000), IL-1β rabbit pAb (1:1200) and IL-6 rabbit pAb (1:1200) were provided by Abcam (MA, USA). Mfn2 mouse mAb (1:2,000) and IL-10 mouse mAb (1:2000) were obtained from Santa Cruz Biotechnology (CA, USA). β-actin rabbit pAb (1:2000) was from Bioworld (MN, USA). IFN-β rabbit pAb (1:500) was from OriGene Technology (MD, USA). p-IRF3 rabbit mAb (1:1000), NF-κB p65 rabbit mAb (1:2000) and TNF-α rabbit pAb (1:2000) were provided by Cell Signaling Technology (MA, USA). MAVS rabbit pAb (1:500) and STING rabbit pAb (1:500) antibodies were obtained from Proteintech Group (IL, USA).

Cells were treated in α-MEM containing 2 μg/mL Hlb for the indicated minutes. To extract total cell protein, cells were harvested after washing with cold PBS and lysed with cold RIPA lysis buffer containing protease and phosphatase inhibitors. The samples were resolved on a 12% SDS-PAGE and electroblotted onto polyvinylidene fluoride (PVDF) membranes. Anti-STAT5A Ab, phospho-STAT5A Ab (BBI Life Sciences), phospho-STAT4 Ab, STAT4 rabbit mAb, NF-κB p65 rabbit mAb, phospho-NF-κB p65 rabbit mAb, p44/42 MAPK rabbit mAb, phospho-p44/42 MAPK rabbit mAb (Cell Signaling Technology), anti-GAPDH, and horseradish peroxidase-conjugated goat antibodies against rabbit IgG and mouse IgG (Jackson ImmunoResearch Laboratories) were used. Finally, the blots were detected using Pierce™ ECL Western Blotting Substrate (Thermo).

The Csf-1-deficient mice were purchased from The Jackson Laboratory, and C57BL/6 mice were purchased from Nanjing University. All mice were housed in the animal facility of Nanjing Medical University.
Sera of SLE patients were collected from The First Affiliated Hospital of Nanjing Medical University. We adopt the criteria of the American College of Rheumatology as the classification of SLE. All patients had SLE disease-activity index scores ranging from 0 to 20.
Protein G agarose was purchased from Millipore. FITC-conjugated CTB, methyl-β-cyclodextrin (MβCD) and β-estradiol 6-(O-carboxy-methyl)oxime: BSA (E2-BSA) were purchased from Sigma-Aldrich. G15 was purchased from TOCRIS bioscience. ICI 182,780 (Fulvestrant), a specific antagonist of nuclear estrogen receptors, was purchased from Santa Cruz Biotechnology.
Anti-CD11b, TNF-α, and MCP-1 antibodies were purchased from Abcam. Anti-phospho-NF-kB p65 rabbit mAb, NF-κB p65 rabbit mAb, and GAPDH rabbit mAb antibodies were purchased from Cell Signaling Technology. Anti-CD3, CD20, GPER1, and CD64 antibodies were purchased from Santa Cruz Biotechnology.

Heat-killed P. acnes-infected HaCaT cells were treated with Ce6-mediated PDT, after which the cells were fixed with 4% paraformaldehyde in PBS for 20 min and permeabilized with 0.5% Trixon X-100 for 15 min. After 1 h incubation with blocking buffer (5% BSA in PBS), the cells were incubated with primary antibodies (NFκB p65 Rabbit mAB) (1:100) (Cell Signaling Technology, Inc., MA, USA) in 0.5% BSA for overnight at 4°C. The cells were washed in PBS for 10 min, done 3 times, and were stained for another 1 h with goat anti-rabbit IgG Texas red (1:1000) (Santa Cruz Biotechnology, Inc., USA). Nuclei were counterstained with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (Bio-Rad, USA). The prepared cells were then observed under a fluorescent microscope and images were recorded.