Islets or INS-1 cells were collected in lysis buffer (Cell Signaling Technology). Protein lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (BioRad). Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated secondary antibodies. Anti-ECHA and anti-SIRT4 was from Abcam. Anti-SIRT3, anti-SIRT5, and anti-rabbit IgG conjugated with HRP were from Cell Signaling Technology. Anti-α-tubulin was from Proteintech. Anti-Flag was from Sigma-Aldrich. Anti-acetyllysine was from PTM Biolab. Immunoprecipitation was performed by incubating protein lysates with FLAG M2 Affinity Gel (Sigma) or ECHA antibody for 2 h and then with Protein A/G PLUS-Agarose (Santa-Cruz) overnight at 4 °C. The binding complexes were washed and then eluted with loading buffer. Standard western blotting was followed using anti-acetyllysine antibody for acetylation analysis.
Anti sirt5
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SIRT5 is a primary antibody that recognizes the SIRT5 protein. SIRT5 is a member of the sirtuin family of NAD+-dependent deacetylases and plays a role in mitochondrial function and cellular metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Anti sirt3, by Cell Signaling Technology (6 mentions)
Anti sirt6, by Cell Signaling Technology (3 mentions)
Anti sirt4, by Abcam (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti sirt7, by Cell Signaling Technology (2 mentions)
Anti p53, by Santa Cruz Biotechnology (2 mentions)
Anti sirt2, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Flag m2 affinity gel, by Merck Group (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti α tubulin, by Proteintech (1 mentions)
Anti phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Anti sirt1, by Cell Signaling Technology (1 mentions)
Anti p21, by Merck Group (1 mentions)
Anti phospho sirt 1 ser47, by Cell Signaling Technology (1 mentions)
Anti poly adp ribose polymerase parp, by BD (1 mentions)
8 protocols using anti sirt5
1
Western Blot Analysis of Acetylation
2
Protein Expression Profiling of Rapha Myr-Treated Cells
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Whole cell protein of untreated and 24 h Rapha Myr® extract (0.5–1.25–2.5% v/v)-treated cells were prepared according to Laemmli (1970) and submitted to Western blot analysis, performed according to Grabowska et al., 2016 [72 (link)]. The primary antibodies used were: anti-integrin α5 (1:1000) (Immunological Sciences, Rome, Italy), anti-GAPDH (1:50,000) (Millipore, Darmstadt, Germany), anti-Poly (ADP-ribose)polymerase (PARP, 1:1000) (BD Biosciences, San Jose, CA, USA), anti-γH2AX Ser139 (1:1000) (Abcam, Cambridge, UK), anti-p53 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (1:500) (Sigma-Aldrich, St. Louis, MO, USA), anti-phospho-p53 Ser15 (1:250), anti-sirt 1 (1:250), anti-phospho-sirt 1 Ser47 (1:250), anti-sirt 3 (1:500), anti-sirt 5 (1:500), anti-sirt 6 (1:1000) and anti-sirt 7 (1:250) (Cell Signalling Technology, Denvers, CO, USA). Each protein target was detected by using specific secondary horseradish peroxidase-conjugated antibodies (1:2000) (Dako, Glostrup, Denmark) and an ECL system (Thermo Scientific, Rockford, IL, USA). The expression level of proteins was measured by densitometric analysis using the software Image J and GAPDH was chosen as the reference protein.
3
Profiling Acetylated Mitochondrial Proteins
4
Histone Extraction and Protein Analysis
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Cells and tissues are lysed and homogenized with RIPA buffer (Thermo Fisher Scientific, Cat. #89900) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Cat. # PI78447), 5 M trichostatin A (Cayman Chem, Cat. #89730), and 5 mM nicotinamide (Sigma-Aldrich, Cat. #72340). Histones were extracted from cells and tissues using a total histone extraction kit (EpiGentek, Cat. #OP-0006). Before SDS-PAGE, protein samples were heated at 70°C for 5 min. Antibodies used are listed as follows: anti-β-tubulin (Cell Signaling Technology, Cat. #2128), anti-SIRT5 (Cell Signaling Technology, Cat. #8782), anti-acetyl-lysine (Cell Signaling Technology, Cat. #9814), anti-malonyl-lysine (PTM Biolabs, Cat. #PTM-901), anti-succinyl-lysine (PTM Biolabs, Cat. # PTM-401), anti-glutaryl-lysine (PTM Biolabs, Cat. #PTM-1151), anti-ACC (Cell Signaling Technology, Cat. #3676), anti-phospho-ACC (Ser79) (Cell Signaling Technology, Cat. #3661), anti-H2B (Cell Signaling Technology, Cat. #12364), anti-KAT2A (Cell Signaling Technology, Cat. #3305), anti-HSP60 (Abcam, Cat. #ab59457), anti-Lamin A/C (Cell Signaling Technology, Cat. #2032), anti-COX IV (Abcam, Cat. #ab16056), and anti-GAPDH (Cell Signaling Technology, Cat. #5174). Ponceau S solution (Sigma-Aldrich, Cat. #P7170) was used for protein staining.
5
Protein Extraction and Western Blot Analysis
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Whole cell protein extracts were prepared according to Laemmli (Laemmli 1970 (link)). The primary antibodies used were the following: anti-ATM (1:500), anti-phospho-ATM Ser1981 (1:500), and anti-GAPDH (1:50,000) (Millipore, Warsaw, Poland); anti-p53 (1:500), anti-p21 (1:500), and anti-p16 (1:250) (Santa Cruz Biotechnology, Santa Cruz, USA); anti-phospho-p53 Ser15 (1:250) (Becton Dickinson, Warsaw, Poland); anti-HO-1 (1:1000) (Enzo Life Sciences, Warsaw, Poland); SIRT1 (1:250), phospho-SIRT1 Ser47 (1:250), anti-SIRT2 (1:1000), anti-SIRT3 (1:500), anti-SIRT5 (1:500), anti-SIRT6 (1:1000), anti-SIRT7 (1:250), anti-phospho-p38 Thr180/Tyr182 (1:500), and anti-p38 (1:500) (Cell Signaling Technology, Poznań, Poland); and anti-AGTR1 (1:500) and anti-actin (1:50,000) (Sigma-Aldrich, Poznan, Poland). The respective proteins were detected after incubation with one of the horseradish peroxidase-conjugated secondary antibodies (1:2000) (Dako, Gdynia, Poland), using an ECL system (Thermo Scientific, Warsaw, Poland), according to the manufacturer’s instructions.
6
Immunoblotting of Sirtuin Proteins
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Western blotting was conducted as previously described [45 , 46 ]. The primary antibodies used are as follows: anti‐Sirt3 (#5490; Cell Signaling Technology, Boston, MA, USA), anti‐Sirt4 (ab124521; Abcam, Cambridge, MA), anti‐Sirt5 (#8782; Cell Signaling Technology), anti‐α‐tubulin (Beyotime, Shanghai, China), anti‐hexokinase II (ab104836; Abcam), anti‐PDHE1α (ab168379; Abcam), anti‐SDH (ab14714; Abcam), anti‐COX‐IV (ab33985; Abcam), pan‐succinyl‐lysine (PTM‐401), pan‐malonyl‐lysine (PTM‐901), pan‐glutaryl‐lysine (PTM‐1151), pan‐acetyl‐lysine (#9441; Cell Signaling Technology) and Flag (4110‐20; Shanghai Genomics, Shanghai, China). The antibodies were purchased commercially.
7
Immunohistochemical Analysis of SIRT5 Expression
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Tissues were fixed in formalin, embedded in paraffin, and sectioned before being mounted on slides, which were then subjected to deparaffinization and rehydration. Then, the slides were microwaved for 30 minutes in 0.01 mol/L sodium citrate buffer (pH 6.0). After antigen retrieval and pre-incubation with 10% normal goat serum, anti-SIRT5 (1: 400; Cell Signaling Technology, Danvers, MA, USA) was employed at 4°C overnight. These slides were stained using a VECTSDTSIN Elite ABC Kit (Vector Laboratories, Maravai LifeSciences, San Diego, CA, USA) and counterstained with hematoxylin. The intensity of staining was scored as follows: 0, no staining; 1, weak, light yellow; 2, moderate, yellow-brown; and 3, strong, brown. In addition, the proportion of positive cells was categorized as follows: 0, <10%; 1, 10% to 49%; 2, 50% to 74%; and 3, >75%. Then, the staining score was calculated by multiplying the staining intensity score by the proportion of positive cells score. A staining score of 0 or 1 indicated negative SIRT5 expression, whereas higher scores indicated positive expression.
8
Western Blot Analysis of Intestinal Proteins
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Proteins were obtained from Caco-2 cells and intestinal tissues, resolved by electrophoresis, transferred to nitrocellulose filter membranes (HATF00010, Sigma), blocked with 5% skim milk in TBST, incubated with primary antibodies overnight at 4 °C and incubated with a secondary antibody for 1 h at room temperature. Electrogenerated chemiluminescence (ECL, P10300, NcmBiotech) produced the corresponding protein color. GAPDH was used as a control for whole homogenates. The results were analyzed for gray values using Fiji software. The primary antibodies used were anti-GAPDH (5174, Cell Signaling Technology), anti-Occludin (91,131, Cell Signaling Technology), anti-SIRT3 (5490, Cell Signaling Technology), anti-GPX4 (59,735, Cell Signaling Technology), anti-FTH1 (4393, Cell Signaling Technology), anti-ACSL4 (ab155282, Abcam), anti-ZO-1 (13,663, Cell Signaling Technology), anti-SIRT4(69,786, Cell Signaling Technology), anti-SIRT5(8782, Cell Signaling Technology), anti-SOD2 (24127-1-AP, Proteintech), anti-catalase (24127-1-AP, Proteintech) and anti-FoxO3a (10894-1-AP, Proteintech), and secondary antibody (7074, Cell Signaling Technology).
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