The HEK293T cell line was a gift from the Zhao lab of Fudan University (Shanghai, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Invitrogen), penicillin (Invitrogen) (100 U/ml), and streptomycin (Invitrogen) (100 U/ml). Full-length PEPCK1 (wildtype, 3K/R and 3K/Q) and SIRT2 (wildtype and H187Y) plasmids were also gifts from the Zhao lab of Fudan University (Shanghai, China). Plasmids were cloned to Flag- or HA-tagged destination vectors according to different needs. Point mutations for PEPCK1 and SIRT2 were generated by site-directed mutagenesis. Antibodies against Flag (Sigma, St. Louis., MO, USA), HA (Santa Cruz, Dallas, TX, USA), PEPCK1 (Santa Cruz), SIRT2 (Sigma), α-Tubulin (CST, Danvers, MA, USA), acetylated α-Tubulin (Abcam, Cambridge, UK), and β-actin (Sigma) were all purchased, and the polyclonal antibody against acetyl-lysine was a gift from the Zhao lab. Trichostatin A (CST), nicotinamide (Sigma), sirtinol (Selleck, Houston, TX, USA), MG132 (Sigma), EX527 (Selleck), SRT1720 (Selleck), and CHX (Sigma) were all purchased. Control and siSIRT2 adenovirus were purchased (Vector Biolabs, Malvern, PA, USA).
Acetylated α tubulin
Manufactured by Abcam
Sourced in United States
Acetylated α-tubulin is a component of the cytoskeleton in eukaryotic cells. It is a post-translationally modified form of the α-tubulin protein, where the lysine 40 residue is acetylated. This modification is thought to play a role in the stability and function of microtubules, which are essential structures for cell shape, motility, and intracellular transport.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (4 mentions)
Pepck1, by Santa Cruz Biotechnology (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
Sirt2, by Merck Group (2 mentions)
Nicotinamide, by Merck Group (1 mentions)
Srt1720, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Sirtinol, by Selleck Chemicals (1 mentions)
Ex527, by Selleck Chemicals (1 mentions)
Flk 1, by Santa Cruz Biotechnology (1 mentions)
Lsm 700, by Zeiss (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Zen software, by Zeiss (1 mentions)
β catenin, by Santa Cruz Biotechnology (1 mentions)
Jasplakinolide, by Abcam (1 mentions)
γ tubulin, by Merck Group (1 mentions)
7 protocols using acetylated α tubulin
1
Molecular Mechanisms of PEPCK1 Regulation
2
Immunofluorescence Staining of Cell Cultures
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Cells were grown on four-well plastic dishes. After incubation, the cells were washed twice with PBS and then fixed with 4 % paraformaldehyde in PBS for 30 min at room temperature. The cells were washed again with PBS and then permeabilized for 30 min in PBS containing 0.2 % Triton. Next, the cells were blocked in PBS containing 10 % goat serum and incubated for 1 h with CD90, CD31, vascular endothelial growth factor (VEGF) receptor 1 (Flk-1), β-catenin (Santa Cruz Biotechnology, 1:200), and acetylated α-tubulin (Abcam, Cambridge, MA, USA, 1:200). The cells were washed again three times for 10 min with PBS and incubated with a FITC (fluorescein isothiocyanate)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA, 1:500) for 1 h. Finally, the cells were treated with DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) to stain nuclei for 2 min and then mounted on slides. Photographs of the cells were acquired by using an immunofluorescence microscope (Carl Zeiss, Oberkochen, Germany, LSM700). All images were acquired by using an excitation filter with a reflected light fluorescence microscope and transferred to a computer equipped with ZEN software (Carl Zeiss).
3
Mammalian Expression Plasmids for Cytoskeleton-Associated Proteins
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Mammalian expression plasmids for GFP-MLL2-SET, GFP-FMN1, GFP-CDC42EP3, GFP-MYO5B, GFP-CFL2, GFP-SYNPO, Arp2, β-actin, and Rab11a were constructed by insertion of the respective cDNAs into the pEGFP-C1 vector. The DDI/EEV mutant of CFL2 and the Δ752–903 mutant of SYNPO were generated by site-directed mutagenesis and PCR. MLL2 siRNAs (#1: 5′-GCAUGAAGCCGCAGCAAUU-3′; #2: 5′-GUGCCAAGUGCAUGUUCUU-3′), FMN1 siRNAs (#1: 5′-GCAGAAACCUGUCUCCAAA-3′; #2: 5′-GCAAGACAACCAGAAGUAA-3′), and CFL2 siRNAs (#1: 5′-GUUCGACACUUGGAGAGAA-3′; #2: 5′-GGGAGGCAAUGUAGUAGUU-3′) were synthesized by Ribobio. Antibodies against α-tubulin, acetylated α-tubulin, H3K4me1 (Abcam), β-actin (Sigma-Aldrich), γ-tubulin (Sigma-Aldrich), and Rab11a (Invitrogen) were purchased from the indicated sources. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Fluorescein isothiocyanate (FITC)- and rhodamine-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories. FITC-conjugated phalloidin was from Sigma-Aldrich and 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Songon Biotech. MTA, BIX-01294, CK666 (Sigma-Aldrich), cytochalasin D (EMD Millipore), and jasplakinolide (BioVision) were from the indicated sources.
4
Multiparametric Immunofluorescence Characterization of Lung Cell Phenotypes
5
Immunoblotting Antibody Evaluation Protocol
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Chemicals were purchased from Sigma (St Louis, MO, USA) unless indicated otherwise. Antibodies against GAPDH and α-tubulin were purchased from Sigma. The mouse anti-Cyclin B1 and the rabbit anti-ERLIN2 antibodies were from Cell Signaling (Beverly, MA, USA). Antibodies against V5 were from Invitrogen (Grand Island, NY, USA). NeuN antibody was from EMD Millipore (Billerica, MA, USA). Acetylated-α-tubulin was from Abcam (Cambridge, MA, USA). The rabbit anti-Cyclin B1, mouse anti-HA and mouse anti-Cdk1 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The mouse anti-Cdc27 antibody was from BD transduction Laboratary (San Jose, CA, USA). Bortezomib (B-1408) was from LC Laboratories (Woburn, MA, USA).
6
Regulation of PEPCK1 by SIRT2 in Hepatocytes
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Human liver cell lines (HepG2, Huh-7, Hep3B, 7721, and Chang's) were donated by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and the Zhao lab of Fudan University (Shanghai, China). Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (Invitrogen), penicillin (Invitrogen) (100 U/ml), and streptomycin (Invitrogen) (100 U/ml). Full-length PEPCK1 (wild type, 3K/R and 3K/Q) and SIRT2 (wild type and H187Y) plasmids were also donated from the Zhao lab of Fudan University (Shanghai, China). Antibodies against Flag (Sigma, St. Louis., MO), PEPCK1 (Santa Cruz), SIRT2 (Sigma), α-tubulin (CST, Danvers, MA), acetylated α-tubulin (Abcam, Cambridge, UK), and β-actin (Sigma) were all purchased, and the polyclonal antibody against acetyl-lysine was a donation from the Zhao lab. Selisistat (Selleck, Houston, TX), MG132 (Sigma), salermide (Sigma), and CHX (Sigma) were all purchased. Control and siSIRT2 adenovirus were purchased (Vector Biolabs, Malvern, PA).
7
Protein Expression and Modification Analysis
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Cells were lysed with 0.5% NP40 lysis buffer and proteins were blotted following standard protocol. Signals were probed using the chemiluminescence ECL plus reagent (Thermo, Grand Island, NY, USA) and detected using a Typhoon FLA9500 scanner (GE, Fairfield, CT, USA). Primary antibodies were as follows: HDAC1 (abcam, Cambridge, UK), HDAC2 (abcam), HDAC3 (abcam), HDAC4 (CST, Danvers, MA, USA), HDAC5 (Santa Cruz), HDAC6 (CST), HDAC7 (abcam), cleaved caspase-3 (Antibody Revolution, San Diego, CA, USA), cleaved PARP (CST), β-actin (Sigma), acetyl-histone H3 (Millipore), acetylated α-tubulin (abcam), α-tubulin (CST), phospho-p70 S6 kinase (CST) and p70 S6 kinase (CST). LC3, P53, PUMA, HA, and anti-histone H3 antibodies were kindly provided by the Zhao lab of Fudan University (Shanghai, China).
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