Cells were washed with phosphate buffered saline (PBS) and then lysed on ice in RIPA buffer (Sigma Aldrich) containing Phosphatase inhibitor cocktail 2 (Sigma Aldrich), Complete protease inhibitor cocktail (Roche), 100mM NaF, 4mM Na3VO4, 1mM phenylmethylsulfonylfluoride (PMSF), 1mM dithiothreitol (DTT) and 100U/ml benzonase (Sigma Aldrich). Samples were resolved by SDS-PAGE on 4–12% Novex Bis-Tris precast gels (Thermo Fisher Scientific) and analyzed by western blotting using the following antibodies: fibronectin sc-6952 (Santa Cruz Biotechnology), YAP #14074, TAZ #4883, Smad2 #5339, tubulin #2148, phospho-YAP (Ser127) #13008 (Cell Signaling Technologies), αSMA ab5694, GAPDH ab9485, Smad3 ab40854, phopsho-Smad3 (S423+S425) ab52903 (Abcam), collagen type I OAMA03716, collagen type III OASB02204 (Aviva), vimentin #M7020 (Dako Cytomation) and HRP-coupled secondary antibodies (GE Life Sciences, Thermo Fisher Scientific). Membranes were treated with Western Lightning Enhanced Chemiluminescence Substrate (Perkin Elmer), and the chemiluminescence signal was recorded by the chemiluminescence reader Fusion FX6 (Vilber-Lourmat). Signals were quantified using densitometric analysis in ImageJ software.
Tubulin 2148
Manufactured by Cell Signaling Technology
Tubulin (#2148) is a purified protein product from Cell Signaling Technology. It is a major component of the cytoskeleton and plays a critical role in cellular structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Radio immunoprecipitation assay ripa, by Merck Group (3 mentions)
Sox2 ab171380, by Abcam (2 mentions)
Hrp coupled secondary antibodies, by GE Healthcare (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Benzonase, by Merck Group (1 mentions)
Gapdh ab9485, by Abcam (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Fibronectin sc 6952, by Santa Cruz Biotechnology (1 mentions)
Yap 14074, by Cell Signaling Technology (1 mentions)
Taz 4883, by Cell Signaling Technology (1 mentions)
Smad2 5339, by Cell Signaling Technology (1 mentions)
Vimentin m7020, by Agilent Technologies (1 mentions)
Western lightning enhanced chemiluminescence substrate, by PerkinElmer (1 mentions)
Fusion fx6, by Vilber (1 mentions)
Actin a5441, by Merck Group (1 mentions)
Gapdh mab374, by Merck Group (1 mentions)
Ab27053, by Abcam (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ab76008, by Abcam (1 mentions)
5 protocols using tubulin 2148
1
Western Blot Analysis of Fibrosis Markers
2
Cell and Zebrafish Lysis Protocol
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Cells were lysed in HR lysis buffer
(50 mM Tris, 5 mM MgCl2, 250 mM sucrose, and 2 mM DTT at
pH 7.4) with protease inhibitor cocktail for 10 min on ice. The lysates
were sonicated using 10 cycles of 30 s pulse on, 30 s pulse off. Twenty
Zebrafish embryos in each group were lysed in Laemmli buffer and boiled
for 5 min. The lysates were centrifuged at maximun speed for 20 min
at 4 °C. Thereafter, protein concentrations were measured using
the DC protein assay (500-0111, Bio-Rad), and equal amounts of proteins
were used for each condition that was analyzed by WB with following
antibodies: UCHL1 (ab27053, Abcam) for cells, UCHL1 (HPA005993, ATLAS)
for zebrafish embryos, PARK7 (ab76008, Abcam) for zebrafish embryos,
Tubulin (2148, Cell Signaling) for cells and zebrafish embryos, GAPDH
(MAB374, Millipore) for cells, and Actin (A5441, Sigma-Aldrich) for
cells.
(50 mM Tris, 5 mM MgCl2, 250 mM sucrose, and 2 mM DTT at
pH 7.4) with protease inhibitor cocktail for 10 min on ice. The lysates
were sonicated using 10 cycles of 30 s pulse on, 30 s pulse off. Twenty
Zebrafish embryos in each group were lysed in Laemmli buffer and boiled
for 5 min. The lysates were centrifuged at maximun speed for 20 min
at 4 °C. Thereafter, protein concentrations were measured using
the DC protein assay (500-0111, Bio-Rad), and equal amounts of proteins
were used for each condition that was analyzed by WB with following
antibodies: UCHL1 (ab27053, Abcam) for cells, UCHL1 (HPA005993, ATLAS)
for zebrafish embryos, PARK7 (ab76008, Abcam) for zebrafish embryos,
Tubulin (2148, Cell Signaling) for cells and zebrafish embryos, GAPDH
(MAB374, Millipore) for cells, and Actin (A5441, Sigma-Aldrich) for
cells.
3
Molecular Profiling of LSCC Cancer Stem Cells
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For research use, the LSCC samples were collected from the Department of Oncology, Shanghai Chest Hospital. Written consent forms were obtained from every LSCC patient. The Institute Research Ethics Committee of Shanghai Chest Hospital approved all protocols.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
4
Protein Expression Analysis in LSCC
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For research use, the LSCC samples were collected from the Department of Oncology, Shanghai Chest Hospital. Written consent forms were obtained from every LSCC patient. The Institute Research Ethics Committee of Shanghai Chest Hospital approved all protocols.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
5
Molecular Profiling of LSCC Cancer Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For research use, the LSCC samples were collected from the Department of Oncology, Shanghai Chest Hospital. Written consent forms were obtained from every LSCC patient. The Institute Research Ethics Committee of Shanghai Chest Hospital approved all protocols.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
Western Blotting YAP1(#4912), LATS1(#9153), p-YAP1(#4911), Vimentin(#12020), Snail(#3879) and Tubulin(#2148) antibodies were all from Cell Signaling Technology (CST, Danvers, MA). OCT4 (ab19875), CD133 (ab19898) and SOX2 (ab171380) were from Abcam. Protein was obtained from cells by using Radio-Immunoprecipitation Assay (RIPA) (Sigma-Aldrich). Equal quantities of protein lysates were loaded on the Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis gels (SDS-PAGE gels), and gel electrophoresis was applied to transfer the protein onto a nitrocellulose membrane. The protein strips were ultimately detected by enhanced chemiluminescence (Thermo Fisher Scienti c) and captured using the Odyssey Infrared Imaging Analysis Software.
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