All immunoblots were carried out according to standard procedures. For the indicated assessments, the cells were lysed in standard buffers. Lysates with equal amounts of protein content were resolved using SDS–PAGE and electrotransferred to PVDF membrane (Bio-Rad, Hercules, CA, USA). Blots were probed with antibodies against IDH1 R132H mutant (MBL Life Sciences, Woburn, MA, USA), acetylated α-tubulin (Santa Cruz Biotechnology, Dallas, TX, USA), acetylated Histone-H4 (Santa Cruz), eIF5 (Santa Cruz), and GAPDH (Millipore Sigma, Burlington, MA, USA), according to the manufacturer’s recommendations. The appropriate horseradish peroxidase-conjugated secondary antibodies and reagents were used for chemiluminescent detection, according to manufacturers’ recommendations. After the application of the chemiluminescent substrate, the blots were exposed to standard X-ray film for appropriate lengths of time.
Acetylated α tubulin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Acetylated α-tubulin is a component of the cytoskeleton, specifically the microtubules, which play a crucial role in various cellular processes. This post-translationally modified form of α-tubulin is involved in the stabilization of microtubules and the regulation of their dynamics.
Automatically generated - may contain errors
Lab products found in correlation
P47phox, by Santa Cruz Biotechnology (2 mentions)
P22phox, by Bioneer (2 mentions)
P47phox, by Bioneer (2 mentions)
Hdac6, by Bioneer (2 mentions)
Cxcl8, by Bioneer (2 mentions)
Cxcl10, by Bioneer (2 mentions)
β actin, by Bioneer (2 mentions)
Oligonucleotide primers, by Bioneer (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Cep164, by Proteintech (2 mentions)
Centrin, by Merck Group (2 mentions)
Cep164, by Santa Cruz Biotechnology (2 mentions)
Colchicine, by Merck Group (2 mentions)
Thapsigargin, by Thermo Fisher Scientific (2 mentions)
Nocodazole, by Merck Group (2 mentions)
Chloroquine, by Merck Group (2 mentions)
Bapta, by Thermo Fisher Scientific (2 mentions)
Paclitaxel, by Avantor (2 mentions)
Vivit, by Cayman Chemical (2 mentions)
Mdia1, by Cell Signaling Technology (2 mentions)
16 protocols using acetylated α tubulin
1
Immunoblot Analysis of Protein Modifications
2
Investigating Oxidative Stress Signaling
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Primary antibodies against gp91phox/NOX2, p47phox, acetylated α-tubulin, p65, IkB, c-fos, and c-jun were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Total-ERK, total-p38, total-JNK, phosphor-ERK, phosphor-p38, phosphor-JNK, β-actin, and HDAC6 were purchased from Cell Signaling Technology (Beverly, MA, U.S.A.). Hindsiipropane B was synthesized from commercially available starting materials with the Wittig-Horner reaction, Claisen-Schmidt condensation and hydrogenation as key steps (14 (link)). 10 mM stock solution of hindsiipropane B was prepared in dimethyl sulfoxide. Oligonucleotide primers (HDAC6, CCL2, CXCL8, CXCL10, Nox2, p22phox, p47phox, and β-actin) (Bioneer, Seoul, Korea) were obtained from commercially.
3
Molecular Mechanisms of Oxidative Stress Regulation
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N-acetyl cysteine (NAC), diphenylene iodonium (DPI), apocynin (APO), and tubastatin A (TBA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dihydroethidium (DHE) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Primary antibodies against gp91phox/NOX2, p47phox, acetylated α-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and those against HDAC6 and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 3000 transfection reagent was purchased from Invitrogen (Carlsbad, CA, USA). A luciferase assay kit was purchased from Promega (Madison, WI, USA). Oligonucleotide primers (HDAC6, CCL2, CXCL8, CXCL10, Nox2, p22phox, p47phox, and β-actin) and HDAC6 siRNA were obtained from Bioneer (Seoul, Korea).
4
Western Blot Analysis of LV Myocardial Samples
5
Quantitative Super-Resolution Imaging of Acetylated Microtubules
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Transfected cells were seeded into 6-well dishes containing 5 mm round coverslips and 2 mL fresh media treated with chloroquine after 24 hours, followed by fixation for 10 minutes in a 1:1 ratio of 4% Paraformaldehyde: DMEM at 37°C. Cells were blocked using 3% donkey serum in PBS, followed by probing the required primary antibody, i.e. acetylated α-tubulin (Santa Cruz, 23950) overnight at 4°C. Next, cells were incubated with a secondary antibody, Alexa Fluor 568 (ThermoFisher, A-10042) for 50 minutes. Coverslips were then washed 3x5 minutes with PBS and using Dako® fluorescent mounting media. For SR-SIM analysis of GFP-Tau transfected cells, thin (0.1 μm) z-stacks of high-resolution (1024x1024 pixels) image frames were collected by utilizing an alpha Plan-Apochromat 60x/1.4 oil immersion DIC M27 ELYRA objective on an ELYRA PS.1 system (Carl Zeiss Microimaging, Germany) equipped with a 488nm laser (100mW) in 5 rotations, 561nm laser (100mW) in 5 rotations and an Andor EM-CCD camera (iXon DU 885). Images were reconstructed using Zeiss Zen Black Software (2012) based on a structured illumination algorithm [22 ]. Colocalization analysis of these reconstructed super-resolution images using both the 2D and 3D VR-assisted 3D ROI selection systems.
6
SARS-CoV-2 Immunostaining of Human Tissues
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The fixed paraffin-embedded ex-vivo cultures of human tissues were stained with cell type-specific markers (MUC5AC, ThermoFisher, Waltham, MA, USA; acetylated α-tubulin, Santa Cruz, Dallas, TX, USA; CC10, Protein-tech, Rosemont, IL, USA; p63a, Cell Signaling Technology, Danvers, MA, USA; pan cytokeratin AE1/AE3 and CD68, Dako, Agilent Technologies, Santa Clara, CA, USA), SARS-CoV-2 and SARS-CoV nucleoprotein (4D11),11 (link) MERS-CoV nucleoprotein,7 (link), 12 (link) and influenza nucleoprotein (HB65, EVL anti-influenza nucleoprotein, subtype A).9 , 13 (link) Methods of immunohistochemistry staining are detailed in the appendix (p 3) .
7
Immunofluorescence Staining of Acetylated Tubulin
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SCP-1 cells were fixed with a 4% formaldehyde solution for 10 min at room temperature, then incubated with 0.2% Triton-X-100 for 10 min, and subsequently with 2% formaldehyde for 10 min. After that, cells were blocked with 5% bovine serum albumin (BSA, Carl Roth, Darmstadt, Germany) for 1 h. Then, cells were incubated with primary antibody solution (acetylated α-tubulin, 1:100, Santa Cruz, Heidelberg, Germany) overnight at 4 °C. The next day, cells were washed with PBS and incubated with Alexa-488 labeled secondary antibody (1:2000, Invitrogen, Karlsruhe, Germany) and Hoechst 33,342 (2 ng/mL) for 2 h. Fluorescent images were taken with a fluorescence microscope (200- or 400-fold magnification) and analyzed with the ImageJ software [13 (link)].
8
Immunofluorescence Analysis of Hedgehog Signaling
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Immunofluorescence was performed on chamber slides (Nalge Nunc International, Naperville, IL). After rinsed in PBS, samples (cells or limb bud tissues) were fixed in ice-cold methanol and permeabilized with 0.1% Triton X-100 in PBS (PBST). After incubation with blocking buffer for 30 min at room temperature, samples were incubated with primary antibodies against Smo (ab236465, abcam), Gli1 (sc-20687, Santa Cruz), pVav2 (ab86695, abcam), Vav2 (sc-271442, Santa Cruz), acetylated-αTubulin (sc-23950, Santa Cruz), KIF3A (sc-376680, Santa Cruz), IFT88 (sc-84318, Santa Cruz), pPAK1 (#2601, Cell Signaling Technology), PAK1 (#2602, Cell Signaling Technology), Arl13b (17711-1-AP, Proteintech) or β-actin (sc-47778, Santa Cruz) overnight at 4 °C. After washing with PBST, samples were further incubated with Alexa 488-conjugated or 555-conjugated secondary antibody (Life Technology). The nuclei were counterstained with 6'-diamidino-2-phenylindole (DAPI), and immunostaining was analyzed by a laser scanning microscope (Zeiss).
9
Immunofluorescence Assay for Peroxisome Analysis
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Briefly, cells were fixed in 4% paraformaldehyde; blocked and permeabilized with 3% bovine serum albumin (Sigma) with 0.1% Triton X-100; incubated with antibodies against microtubules (1:1000, acetylated α-tubulin, Santacruz Biotechnology) and peroxisomes (1:1000, PEX14 [16 (link), 17 (link)]); incubated with secondary antibodies (1:400, Alexa Fluor® 488 and Alexa Fluor® 594, ThermoFisher Scientific) for 30 min and stained with DAPI (1:1000; Life Technologies) to label nucleus. Microtubules were labelled to identify the total cell area.
Cells were visualized and imaged using a Leica SP5 confocal microscope (Leica, Germany) at high magnification of 60×. Resulting images were analysed for peroxisome density and size using image J image analysis software [18 (link)]. For peroxisome density, the total area of the cell and number of peroxisomes in a cell were measured. Peroxisome density was measured and reported as a ratio of number of peroxisomes to the total cell area. The analysis was performed on about 10 cells per cell line. Three controls and one PEX16 patient cell line were used for this analysis. Peroxisome size was also calculated using ImageJ on the same cells.
Cells were visualized and imaged using a Leica SP5 confocal microscope (Leica, Germany) at high magnification of 60×. Resulting images were analysed for peroxisome density and size using image J image analysis software [18 (link)]. For peroxisome density, the total area of the cell and number of peroxisomes in a cell were measured. Peroxisome density was measured and reported as a ratio of number of peroxisomes to the total cell area. The analysis was performed on about 10 cells per cell line. Three controls and one PEX16 patient cell line were used for this analysis. Peroxisome size was also calculated using ImageJ on the same cells.
10
Comprehensive Hedgehog Signaling Analysis
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Antibodies for Taz (sc-48,805), Gli1 (sc-20,687), Gli2 (sc-271,786), Gli3 (sc-74,478), c-myc (sc-40), PKAc (sc-903), p-PKA (T198, sc-32,968), GSK3β (sc-9166), CK1α (sc-74,582), acetylated-α-tubulin (sc-23,950), β-actin (sc-69,879), Lamin B (sc-374,015), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, sc-32,233), and rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). YAP (#12395), p-GSK3β (Ser9/21, #8566), Lats1 (#9153), HA (#3724), and Flag (#14793) were from Cell Signaling Technology (Danvers, MA).Gli3 (ab69838, immunogen corresponding to the residues 1–100 of human Gli3), Gli3 (ab181130, immunogen corresponding to the residues 1300–1500 of human Gli3), Smo (ab72130), p-Ser/Thr (ab117253) and Ki67 (ab15580) were purchased from Abcam (Cambridge, UK). Ptc1 (06–1102) was from Millipore (Billerica, MA). The IRDye 680 and 800 second antibodies were from LI-COR Bioscience (Lincoln, NE). GST fusion proteins, including GST-Gli3F, GST-Gli3R, and GST-Taz, were generated as previously described (Einarson et al., 2007). Bioactive Shh recombinant protein (N-Shh) was from PeproTech Inc. (Rocky Hill, NJ), whereas H-89, SB216763, and forskolin were from Sigma (St. Louis, MO).
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