Nf kb p65

Regular Western blotting analysis was conducted as described previously [28 (link)] using the following primary antibodies: anti-light chain (LC)3 (1:1000), Beclin 1 (1:1000), TLR4 (1:1000), MyD88 (1:1000), mTOR (1:1000), phosphorylated (p)-mTOR (1:1000), p38 (1:1000), (p)-p38 (1:800), NF-kB p65 (1:1000), Akt (1:1000), p-Akt (1:2000), p-PI3K (1:1000), and PI3K (1:800) (Cell Signaling Technology, Inc., Danvers, MA, USA). Densitometry analysis was performed using the ImageJ Gel Analysis tool (NIH, Bethesda, MD, USA).

The total DNA/RNA/Protein Kit (R6734, Omega Bio-tek, Norcross, GA, USA) was utilized to extract total protein from hippocampal tissue according to the manufacturer’s instructions. Western blot was conducted for CREB (#9197, Cell signaling technology, USA), p-CREB (#9198, Cell signaling technology, USA), BDNF (ab108319, Abcam, UK), AKT (#9272, Cell signaling technology, USA), p-AKT (#4060, Cell signaling technology, USA), PSD95 (#3450, Cell signaling technology, USA), GABAA (GB113973, Servicebio, Wuhan, China), Synapsin-1 (SYN, #5297, Cell signaling technology, USA), Bax (#2772, Cell signaling technology, USA), Bcl-2 (#3498, Cell signaling technology, USA), Caspase-3 (#9662, Cell signaling technology, USA), Cleaved-caspase 3 (#9661, Cell signaling technology), NF-kB (P65) (#8242, Cell signaling technology, USA), NLRP3 (sc-134306, Santa Cruz, CA, USA), Caspase 9 (#9504, Cell signaling technology), Caspase 1 (#3866, Cell signaling technology), β-actin (bs-10966R, Bioss, Beijing, China) of hippocampal total protein. The density of protein bands was assessed using a computing densitometer. IL-1β was measured using ELISA kits (Nanjingjiancheng, Nanjing, China) according to the manufacturer’s protocols.

Cell culture materials were purchased from Irvine Scientific (Irvine, CA) or GIBCO (Grand Island, NY) except for skeletal muscle growth medium, which was obtained from Lonza (Walkersville, MD). [3H] 2-deoxyglucose, [¹⁴C]-L-glucose and [³H]-palmitate were obtained from Perkin Elmer (Boston, MA). All other chemicals were reagent grade and purchased from Sigma Chemical (St. Louis, MO), except for AG-1X8 ion exchange resin (Bio-Rad, Richmond, CA). Electrophoresis reagents were from Bio-Rad or Invitrogen (Carlsbad, CA). Primary antibodies were obtained from the following sources: IkBa (catalog #9242), phospho-p44/42 MAPK (#9106), p44/42 MAPK (#4695), phospho-p38 MAPK (#9216), p38 MAPK (#9212), phospho-NF-kB p65 (#13346), NF-kB p65 (#8242) (Cell Signaling Technology, Beverly, MA), phospho-JNK (#sc-6254), JNK (#sc-571), MyoD (#sc-760) (Santa Cruz Biotechnology, Santa Cruz, CA), b-actin (#NB600-503) (Novus, Littleton, CO). Fluorescently labeled secondary antibodies and blocking buffer were obtained from Licor (Lincoln, NE).

A capillary-based immunoassay was performed using the Size Separation Master Kit (SM-W004, ProteinSimple, San Francisco, CA, USA) with Split Buffer (12–230 kDa) by the Wes (WS3312, ProteinSimple, San Francisco, CA, USA) [47 (link),48 (link),49 (link),50 (link)]. Protein expression was measured by chemiluminescence and quantified as area under the curve using the Compass for Simple Western program (Compass for SW 5.0.1, ProteinSimple, San Francisco, CA, USA). Proteins were detected with the following primary antibodies: NF-kB p65 (8242, cell signaling); HMGB1 (D3E5) (6893, Cell signaling, Boston, MA, USA); NLRP3 (D4D8T) (15101S, cell signaling, Boston, MA, USA); ASC/TMS1 (D2W8U) (67924, Cell signaling, Boston, MA, USA); GAPDH (5174, Cell signaling, Boston, MA, USA).

At the end of the experiments silicone membranes with confluent monolayers were stained with specific antibodies for NFKB p50/p105 (Santacruz, California), NFKB p65 (Cell Signaling, Massachusetts) and CDH5 and visualized using fluorescence microscopy. Silicone membranes were washed twice with PBS and fixed in 1% PFA and 4% PFA for NFKB p65 for 10 min, cut into sections, and permeabilized with Triton X-100 in PBS for 10 min. After permeabilization samples were blocked in 10% BSA and 0.1% Triton X-100 in PBS (NFKB and CDH5) for 1h. After washing with 0.1% Triton X-100 in PBS, the silicone sheets were incubated with polyclonal rabbit anti- NFKB, and CDH5 primary antibodies overnight at 4°C followed by washing with 0.1% Triton X-100 in PBS. Samples were subjected to secondary antibody Alexa Fluor 488 donkey anti-rabbit (1:500 to 1:200; Invitrogen) secondary antibody for 1.5 h for CDH5 and with secondary antibody Alexa Fluor 488 donkey anti-goat (1:100 to 400; Santa Cruz) for 1.5h for NFKB. Samples were washed again with 0.1% Triton X-100 in PBS and mounted with vectashield mounting media with DAPI on glass slides with cover slips in contact with cells. These slides were imaged using a Nikon Eclipse TE2000-E inverted fluorescence microscope with a Photometrics Cascade 650 camera (Roper Scientific) and MetaVue 6.2r2 imaging software (Universal Imaging).

We used the following antibodies: Dclk1, Lgr5, Bmi1, Hes1, Tcf4, Cox1, Cox2, EpCam, CD45, CD31 (all from Abcam, Cambridge, MA), CXCL1, CyclinD1, cMYC, β − catenin (Santa Cruz Biotechnology, USA), Notch1, NfkB-p65, CyclinD1, Ras, β-actin (Cell Signaling, Danvers, MA, USA), anti-rabbit IgG, anti-mouse IgG, anti-goat IgG (Jackson ImmunoResearch, West Grove, PA, USA), Alexa Fluor® 488 donkey anti-rabbit IgG, and Alexa Fluor® 568 donkey anti-goat IgG (Invitrogen, USA).

Western blot analyses were performed as previously described.8 Briefly, membranes (nitrocellulose blotting membrane 0.2 µm; GE Healthcare Life Sciences, Freiburg, Germany) were incubated for 2 h with primary antibodies specific for phospho NF‐κB p65 (Ser536; 1:1,000; rabbit monoclonal; Cell Signaling, Danvers, MA), NF‐kB p65 (1:1,000; mouse monoclonal; Cell Signaling), and β‐actin (1:5,000; mouse monoclonal; Cell Signaling). Thereafter, membranes were incubated for 1 h with a solution containing IRDye 800CW goat anti‐rabbit IgG (1:15,000; LI‐COR, Bad Homburg, Germany) and IRDye 680LT goat anti‐mouse IgG (1:15,000; LI‐COR, Bad Homburg, Germany). Signal intensities were quantified using the Odyssey Imager CLx (LI‐COR, Bad Homburg, Germany). The ratios between levels of phospho‐p65 and total p65 (both normalized to β‐actin) were calculated and depicted as absolute signal intensities.