The reprogrammed cell lines were qualitatively evaluated by using a phase-contrast microscope and manually assigned to one of the three morphology groups (stable colony, unstable class 1, and unstable class 2). Representative lines for each colony morphology group were imaged using a Nikon TE2000 with a 10x objective. Immunocytochemistry analysis was performed on a representative line for each colony morphology group. Cells were cultured on glass coverslips and fixed in 2% PFA for 15 min. The immunofluorescence protocol was performed following the guidelines provided by the suppliers. The following antibodies were used: mouse anti-human α-tubulin (1/100, cat# T5168, Sigma), rabbit anti-human β-tubulin (1/500, cat# ab32572, Abcam); and the following secondary antibodies were used: donkey anti-rabbit A647 (1/500, Molecular Probes) and donkey anti-mouse A594 (1/500, Molecular Probes). The nuclei were stained with DAPI (cat# D1306, Molecular Probes). The samples were mounted in ProLong Diamond Antifade Mountant Media (cat# P36970, Life Technologies). The expression of β-tubulin and α-tubulin was analyzed by using a Leica TCS SP5 confocal microscope with a 40x objective. No specific feature of the original data was obscured, eliminated, or misrepresented.
Mouse anti human α tubulin
Manufactured by Merck Group
Sourced in United States
Mouse anti-human α-tubulin is a primary antibody that binds to the alpha subunit of the tubulin protein, which is a major component of the cytoskeleton in eukaryotic cells. This antibody can be used in various laboratory techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to detect and analyze the presence and distribution of alpha-tubulin.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti rabbit a647, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant media, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Te2000, by Nikon (1 mentions)
Rabbit anti erk, by Cell Signaling Technology (1 mentions)
Mouse anti α tubulin, by Developmental Studies Hybridoma Bank (1 mentions)
Rabbit anti akt, by Cell Signaling Technology (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Rabbit anti myc, by Cell Signaling Technology (1 mentions)
Rat anti ha, by Roche (1 mentions)
Rabbit anti human e cadherin, by Cell Signaling Technology (1 mentions)
Rabbit anti human β catenin, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti human gsk 3β, by Cell Signaling Technology (1 mentions)
Ecl system, by Cytiva (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Las 3000, by Fujifilm (1 mentions)
5 protocols using mouse anti human α tubulin
1
Morphological Evaluation of Reprogrammed Cell Lines
2
Drosophila Protein Antibody Detection
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Rabbit anti‐Drosophila pS6K(T398) and rabbit anti‐Drosophila pAkt(S505) are from PhosphoSolutions (Aurora, CO, USA). Mouse anti‐α‐tubulin (Developmental Studies Hybridoma Bank, #AA4.3), rabbit anti‐ERK (Cell Signaling #4695), rabbit anti‐Akt (Cell Signaling, #4691), rat anti‐HA (Roche, 12CA5), mouse anti‐FLAG M2 (Sigma, F1804), rabbit anti‐myc (Cell Signaling, #2278), rabbit anti‐mammalian pS6K(T389) (Cell Signaling #9205), rabbit anti‐mammalian S6K (Cell Signaling #9202), and mouse anti‐human α‐tubulin (Sigma #T9026) were used in this study. Guinea pig anti‐Drosophila S6K and Tsc2 antibodies were previously described (Hahn et al, 2010 ; Demetriades et al, 2014 ). Antibodies to detect Drosophila eIF4A, Rheb, and RagC were generated by immunizing guinea pigs with full‐length proteins. Anti‐Drosophila‐Raptor and TOR antibodies were generated by immunizing guinea pigs with the peptides ERWQPRARYKKC or PYDPTLQQGGC, respectively, coupled to KLH. Anti‐Drosophila‐Lamp1 antibody was generated by immunizing rabbits with the peptide CARRRSTSRGYMSF coupled to KLH.
3
Western Blot Analysis of Signaling Proteins
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Cells were washed with cold phosphate buffered saline (PBS) and lysed in Pro-Prep solution (Intron, Korea) containing a phosphatase inhibitor cocktail (Sigma, Saint Louis, MO). After lysis, protein quantification was performed using Bradford assays (Biorad, Hercules, CA). Equal amounts of protein were resolved in sodium dodecyl sulfate (SDS)-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Biorad, Hercules, CA). Subsequently, membranes were incubated for 30 minutes in blocking solution (5% nonfat milk), and incubated overnight at 4°C with mouse anti-human IL-32α (Biolegend, San Diego, CA), mouse anti-human α-tubulin (Sigma, Saint Louis, MO), rabbit anti-human β-catenin, rabbit anti-huamn phospho-β-catenin, rabbit anti-human GSK-3β, rabbit anti-human E-cadherin, rabbit anti-human phospho-p44/42 MAPK or rabbit anti-human total p44/42 MAPK (Cell Signaling, Danvers, MA) antibodies (1:1000). After incubation with the primary antibody, membranes were washed three times with PBS containing 0.1% Tween 20 (Merck, Germany) and incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson Laboratory, West Grove, PA). Target proteins were visualized using an ECL system (Amersham Biosciences, UK) and LAS3000 (Fuji Film).
4
Immunoblotting and Immunoprecipitation Analyses
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Immunoblotting was performed exactly as previously described (6 (link), 42 (link)), using the following primary antibodies (1:1,000): rabbit polyclonal anti-phospho FAK Y925 (Cell Signaling, Beverly, MA, USA), rabbit polyclonal anti-FAK (Cell Signaling), rabbit monoclonal anti-human TCF4 (Cell Signaling), mouse anti-E-cadherin (1:2,000; BD Biosciences), mouse anti-human α-Tubulin (Sigma-Aldrich) and appropriate secondary antibodies (diluted 1:5,000): HRP-conjugated goat-anti-mouse or goat-anti-rabbit IgG (Merck-Millipore, Darmstadt, Germany). For immunoprecipitation, cell lysates of Caco2 cells were prepared 72 h after transfection with control or Sdc-1 siRNA as described previously (42 (link)). 0.5 mg protein was incubated with 1:50 dilution of primary antibody (rabbit monoclonal anti-human EGR1, Cell Signaling) at 4°C on a rocker platform overnight. Afterward, the mixture was incubated analogously with 20 μl resuspended protein A/G-PLUS-Agarose. Immunoprecipitates were pelleted by centrifugation (1,000 g, 5 min, 4°C), washed four times with RIPA buffer and boiled in 40 μl SDS sample buffer (5 min). SDS-PAGE, Western blotting, stripping and reprobing were performed as described previously (6 (link)) using 30–60 μg of protein/lane on 7.5– 12% gels.
5
Investigating Cytoskeleton Regulatory Pathways
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