Antibodies for immunoblotting were obtained from the following sources: rabbit anti‐GAPDH (Cell signaling 5174), rabbit anti‐SIRT1 (Cell Signaling 2028), rabbit anti‐phospho‐S6 Ser235/236 (Cell Signaling 4858), and rabbit anti‐S6 (Cell Signaling 2217). Isolated intestine, crypts, or ISCs were lysed in RIPA buffer supplemented with protease Inhibitor and phosphatase inhibitor (Santa Cruz). Proteins extracts were denatured by the addition of SDS loading buffer, boiled and resolved by SDS‐PAGE, and analyzed by immunoblotting with primary antibodies listed above. The band density of all blots was quantified by ImageJ software.
Rabbit anti sirt1
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-SIRT1 is a primary antibody that specifically recognizes the SIRT1 protein. SIRT1 is a NAD-dependent deacetylase enzyme that regulates a variety of cellular processes, including metabolism, apoptosis, and cellular senescence.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin, by Proteintech (2 mentions)
Rabbit anti foxo3a, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Rabbit anti s6, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho s6 ser235 236, by Cell Signaling Technology (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
Ab14745, by Abcam (1 mentions)
Catalase, by Cell Signaling Technology (1 mentions)
Ab110258, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Selleck Chemicals (1 mentions)
Gel image analysis system, by Bio-Rad (1 mentions)
Mtco2, by Abcam (1 mentions)
Uqcrc2, by Abcam (1 mentions)
Ripa buffer, by Selleck Chemicals (1 mentions)
Ascl4, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lysis buffer, by Merck Group (1 mentions)
Rabbit anti sox2, by Cell Signaling Technology (1 mentions)
Rabbit anti taz, by Cell Signaling Technology (1 mentions)
11 protocols using rabbit anti sirt1
1
Immunoblotting with Intestinal Proteins
2
Western Blot Analysis of Cellular Proteins
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The cells were lysed with RIPA buffer containing protease and phosphatase inhibitors (Selleckchem). The protein concentration was tested with a BCA kit, and appropriate amounts of protein were prepared for SDS-PAGE and then transferred to PVDF membrane (Millipore, MA, USA). The membranes were blocked for 1 h with 5% non-fat dry milk and then incubated with rabbit anti-SIRT1 (1:1000; #9475; Cell Signaling Technology Europe, Netherlands); TFAM (1:1000; #8076; Cell Signaling Technology Europe, Netherlands); CATALASE (1:1000; #12,980; Cell Signaling Technology Europe, Netherlands); SOD2 (1:1000; #MA1-106; Invitrogen); UQCRC2 (1:1000; #ab14745; Abcam); MTCO2 (1:1000; #ab110258; Abcam); ASCL4 (1:500; #sc-365230; Santacruz); Actin-b (1:5000, A5441; Sigma) mABs were used as loading controls. The results were imaged using a gel image analysis system (Bio-Rad, California, USA) according to the manufacturer’s instructions.
3
Quantitative Analysis of Lung Protein Expression
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Protein from cells and fetal lungs was collected with lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Samples were electrophoresed in 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with non-fat dried milk diluted in Tris-buffered saline/Tween-20 (TBST) for 1 h. Samples were incubated with primary antibodies including mouse anti-YAP (1:1000; Proteintech, Rosemont, IL, USA), rabbit anti-p-YAP (1:1000; Abcam, Cambridge, UK), rabbit anti-TAZ (1:1000; Cell Signaling, Danvers, MA, USA), rabbit anti-p-TAZ (1:1000; Cell signaling), rabbit anti-FGF10 (1:1000; Abcam), rabbit anti-FGFR2 (1:1000; Abcam), rabbit anti-SOX2 (1:1000; Cell Signaling), rabbit anti-SOX9 (1:1000; Cell Signaling), rabbit anti-SIRT1 (1:1000; Cell Signaling), rabbit anti p-SIRT1 (1:1000; Signalway Antibody, Greenbelt, MD, USA), and mouse anti-β-actin (1:5000; Proteintech) overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, protein bands were detected with the ChemiDoc™ MP Imaging system (Bio-Rad, Hercules, CA, USA) and quantified by Image-Pro software (vers. 4, Media Cybernetics, Rockville, MD, USA). All data were normalized to the control.
4
Western Blot Analysis of Protein Signaling
5
Establishment of SIRT1 Inhibition Model
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BAI (Figure 1(a) ; 98% purity, CAS number 21967-41-9) was bought from Tauto Biotech (Shanghai, China). LPS from Escherichia coli O111:B4 and protease and phosphatase inhibitor tablets were purchased from Sigma-Aldrich (St. Louis, MO, USA). EX-527, also known as Selisistat, was purchased from Selleck (Houston, TX, USA). Rabbit anti-SIRT1, rabbit anti-HMGB1, rabbit antiacetylated lysine, and mouse anti-GFAP were from Cell Signaling Technology (Beverly, MA, USA); mouse anti-TNF-α and mouse anti-HMGB1 were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-IL-1β, goat antimouse IgG H&L (Alexa Fluor® 594), and goat antirabbit IgG H&L (Alexa Fluor® 594/488) were from Abcam (Cambridge, MA, USA); rabbit anti-Iba-1 was from Wako (Rosemont, IL, USA); mouse anti-LaminB1, mouse anti-GAPDH, and mouse anti-β-actin were from Proteintech Group (Wuhan, China). Protein A/G-coupled magnetic beads were from Invitrogen (Carlsbad, CA, USA).
6
EGCG Induces Apoptosis in NPC Cells
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Human NPC cell lines (CNE-2 and 5-8F) were purchased from cell bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1,640 medium and fetal bovine serum (FBS) was purchased from Gibco (Logan, UT, USA). Rabbit anti-caspase 3 (#9,662), Rabbit anti-α-tubulin (#21,44S), Rabbit anti-Apaf-1 (#89,69S), Rabbit anti-caspase 9 (#95,08S), Rabbit anti-Acetyl-p53 (Lys382) (#25,25S), and Rabbit anti-SIRT1 (#2,493) were purchased from Cell Signaling Technology (Boston, MA, USA). EGCG (purity > 95%) was purchased from Sigma Aldrich and dissolved in distilled water at 100 μM, and stored at −20°C until dilution before use. The cell counting kit 8 (CCK8) was purchased from DOJINDO (Japan). The terminal deoxynucleotidyl transferase (TDT)-mediated dUTP nick end labeling (TUNEL) assay kit was purchased from the Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). The BCA protein assay kit was purchased from Thermo Fisher Scientific (Chicago, IL). Super signal chemiluminescent substrate (ECL) was obtained from Thermo Scientific (Waltham, MA, USA).
7
Western Blot Analysis of Brain Tissue
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There were 6 mice conducted western blotting. Brain tissue surrounding the haematoma was homogenized by sonication in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Roche Diagnostics) and centrifuged at 12,000g for 20 min. Proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk in Tris-buffered saline with Tween-20 (TBST), the membranes were incubated with primary antibodies (rabbit anti-claudin-5, 1:1000, Abcam; rat anti-ZO-1, 1:1000, Abcam; rabbit anti-SIRT1, 1:1000, Cell Signaling Technology; rabbit anti-phospho-AMPK(Ser 485), 1:1000, Cell Signaling Technology; rabbit anti-p-AMPK, 1:1000, Cell Signaling Technology; mouse anti-β-actin, 1:2000, Zhongshan Jinqiao, Beijing) overnight at 4 °C. Subsequently, the PVDF membranes were washed three times with TBST and incubated with secondary antibodies (1:5000, Zhongshan Jinqiao, Beijing) at room temperature for 60 min. The blots were visualized using enhanced chemiluminescence (ECL, Cell Signaling Technology) reagents, and band intensities were assessed with Image J software.
8
Retinal Histological Analysis of Liposome Injected Rats
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The model rats were euthanized at 7 days after the intravitreal injection of liposomes. Immediately after the eyeball was removed, it was fixed in 4% paraformaldehyde for 2 h at 4°C, dehydrated overnight with 30% sucrose solution, embedded in an optimal cutting temperature compound (OCT) embedding bottom box (17 × 17 × 5 mm), and stored at −80°C. Slices with thicknesses of 8 μm were created using a frozen slicer at −22°C. The slices were placed on adhesive slides and dried at room temperature for more than 30 min before proceeding to the next step. For immunofluorescent staining, the retinal sections were permeabilized with enhanced immunostaining permeabilization buffer (Beyotime) for 20 min, blocked with blocking buffer (Beyotime), and incubated overnight at 4°C with primary antibodies: Class III β‐TUBULIN (Tuj1 mAb) (Beyotime), Rabbit anti‐IBA1/AIF‐1, rabbit anti‐FOXO3A and rabbit anti‐SIRT1(Cell Signaling Technology). After rinsing three times with PBS, the sections were incubated with secondary antibodies at room temperature for 2 h, rinsed three times with PBS, and counterstained with DAPI for 5 min. The slides were then sealed with cover glass for observation by CLSM.
9
SIRT1 Expression in Intervertebral Disc
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An immunohistochemical staining assay (Boster Biological Technology, China) was used to evaluate the protein deposition and expression level of SIRT1 in human NP tissues and NP cell-encapsulated hydrogels following the manufacturer's protocol. Briefly, the harvested IVDs and hydrogels were sequentially paraformaldehyde-fixed for 24 h, paraffin-embedded and sectioned at 4 mm. Next, the sections were treated with 3% H2O2 for 15 min at room temperature to eliminate endogenous peroxidase activity and were subsequently incubated with 0.125% trypsin for 30 min at 37°C to retrieve the antigen, before being blocked with normal goat serum for 15 min at room temperature. The sections were incubated with rabbit anti-SIRT1 (1:200; Cell Signaling, USA) primary antibodies overnight at 4°C. Then, the sections were incubated with goat anti-rabbit IgG-HRP secondary antibody (1:1000; Proteintech, China) and counterstained with hematoxylin. The resulting sections were photographed under a microscopy (Olympus, Japan). Five fields which distributed throughout the whole section were counted (200×), and the average positive rate was counted and statistically analyzed.
10
Immunofluorescence Staining of Stem Cell Markers
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Immunofluorescence staining was performed as previously reported [27 (link)]. For that purpose, 3 × 103 cells/well were seeded in 24-well plates over rounded glass coverslips and treated as described in the Results section (Section 3.3 , Section 3.4 , Section 3.5 ). Afterwards, cells were fixed in 4% formaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS, blocked with 1% BSA/PBS, and incubated at room temperature for 1 h with the following primary antibodies: rabbit anti-Ki67 (Abcam), mouse anti-NANOG, rabbit anti-Oct4, mouse anti-SOX2, rabbit anti-SIRT1, and rabbit anti-FoxO3a (all from Cell Signaling Technology). Subsequently, samples were washed with PBS and incubated with appropriate FITC-coupled secondary antibodies (Sigma-Aldrich) and 1 ng/mL of the nuclear dye DAPI (Sigma-Aldrich) for 2 h. An epifluorescence microscope (Olympus, Tokyo, Japan) was used for the examination of mounted cell samples.
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