Immunoblotting was performed as described earlier49 (link) using anti-PIMT (1:1000; Santa Cruz Biotechnology, CA, USA) anti-GLUT4 (1:1000; Novus Biologicals, MO, USA), anti-MEF2A (1:1000; Cell Signaling Technology, MA, USA), anti-PGC-1 (1:1000; Santa Cruz Biotechnology, CA, USA),anti-pSer307-IRS1 (1:1000; Cell Signaling Technology, MA, USA),anti-pTyr608/612-IRS (1:1000; Abcam, MA, USA), anti-IRS1 (1:1000; Cell Signaling Technology, MA, USA), anti-pJNK1/2 (1:1000; Cell Signaling Technology, MA, USA), anti-pAkt (Ser473) (1:1000; Cell Signaling Technology, MA, USA), anti-Akt (1:1000; Cell Signaling Technology, MA, USA) ,anti-ERK1/2 (1:1000; Cell Signaling Technology, MA, USA) and anti-pERK1/2 (1:1000; Cell Signaling Technology, MA, USA) using specific antibodies and appropriate HRP-conjugated secondary antibody. Membranes were stripped and re-probed with β-actin (1:1000, ICN Biomedicals Inc., CA, USA) and Tubulin (1:1000, Genscript, NJ, USA) as the protein loading control.
Tubulin
Manufactured by GenScript
Sourced in United States
Tubulin is a globular protein that is the primary component of microtubules, which are essential structures found in the cytoskeleton of eukaryotic cells. Tubulin plays a crucial role in various cellular processes, including cell division, intracellular transport, and cell shape maintenance.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Merck Group (2 mentions)
β catenin, by Thermo Fisher Scientific (2 mentions)
Cell recovery solution, by BD (2 mentions)
Sds polyacrylamide gel, by Bio-Rad (2 mentions)
Ir dye 680 anti mouse or, by LI COR (2 mentions)
Ir dye 800 anti rabbit immunoglobulin g igg secondary antibodies, by LI COR (2 mentions)
Tra2β, by Abcam (2 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Actin, by GenScript (2 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti pimt, by Santa Cruz Biotechnology (1 mentions)
Anti irs1, by Cell Signaling Technology (1 mentions)
Anti glut4, by Novus Biologicals (1 mentions)
Anti pgc 1, by Santa Cruz Biotechnology (1 mentions)
Anti p jnk1 2, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Anti p erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by ICN Biomedicals (1 mentions)
4 protocols using tubulin
1
Immunoblotting Protocol for Metabolic Markers
2
NQO1 Protein Expression Analysis
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Cells were lysed, and total protein concentrations were measured using a BCA protein assay kit (Biosharp, Hefei, China). Based on the molecular weight of NQO1, 10% separating gel and 4% stacking gel were used for electrophoresis at 130 V for 1 h (Bio‐Rad, USA). Samples were then transferred onto membranes at 220 mA for 90 min, and followed by blocking with BSA for 1 h at RT. NQO1 (CST, Shanghai, China) antibody and internal reference Tubulin (GenScript, USA) antibody were diluted and incubated at 4°C overnight. The membrane was washed three times with TBST (5 min/time), and then was incubated with Goat‐anti‐Mouse IgG HRP Conjugate (CWBio, Beijing, China) at RT for 1 h. After that, the membrane was washed three times with TBST again. Finally, the ECL mixture was prepared, and the results were visualized by the chemiluminescence gel imaging system (JS‐1070P: Peiqing, Shanghai, China), and were analyzed using ImageJ software.
3
Protein Expression Analysis in 2D and 3D Cultures
4
Protein Expression Analysis in 2D and 3D Cultures
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3D-grown MCF-10A and MCF-10A MYC-ER cells were washed with PBS (1X) and the Matrigel was dissolved by incubating slides at 4°C in Cell Recovery Solution (BD Biosciences). 2D-grown MDA-MB231 rTTA3 lines and HCC1806-MYC rTTA3 cells were washed with 1xPBS.Cells were lysed in Laemmli buffer (50 mM Tris-HCl pH 6.2, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 3% (w/v) SDS). Equal amounts of total protein were loaded on a stain-free 12% SDS-polyacrylamide gel (Biorad), transferred onto a nitrocellulose membrane (Millipore) and blocked in 5% (w/v) milk in Tween 20-TBST (50 mM Tris pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20). Blots were incubated with TRA2β (Abcam), SRSF1 (CSHL), SRSF3 (MBL), SRSF7 (MBL), c-MYC (Cell Signaling), Actin (GenScript), Tubulin (GenScript) or β-catenin (ThermoFisher) primary antibodies. IR-Dye 680 anti-mouse or IR-Dye 800 anti-rabbit immunoglobulin G (IgG) secondary antibodies (LI-COR) were used for infrared detection and quantification with a ChemiDoc MP Imaging System (Bio-rad).
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