Western Blot Analysis of Cellular Signaling

Cell lysates and western blotting was performed as previously described (Vega-Rubin-de-Celis et al, 2010 (link)), using antibodies from the following sources: S6K1, phospho-S6K1 (T₃₈₉), S6, phospho-S6 (S_235/236), total 4E-BP1, phospho-4E-BP1 (T_37/46), phospho-4E-BP1 (S₆₅) from Cell Signaling; HIF-1α, TSC1 and Menin from Bethyl laboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor from Millipore; Erk1/2, phospho-Erk1/2 (T₁₈₃/Y₁₈₅) and Tubulin from Sigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals; and Grp78 from BD Biosciences. Atp6v1a and Atp6v1b2 antibodies were a generous gift from D Brown (Massachusetts General Hospital, Boston, MA); and Atp6v0a1 and Atp6v0a3 antibodies were kindly provided by V Marshansky (Massachusetts General Hospital, Boston, MA). Tfeb antibodies were developed in collaboration with Bethyl Laboratories.
For the remaining Materials and methods, please see the Supplementary data section.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Peña-Llopis S., Vega-Rubin-de-Celis S., Schwartz J.C., Wolff N.C., Tran T.A., Zou L., Xie X.J., Corey D.R, & Brugarolas J. (2011). Regulation of TFEB and V-ATPases by mTORC1. The EMBO Journal, 30(16), 3242-3258.

Publication 2011

4e bp1 Antibodies Atf4 Atp6v0a1 Atp6v0a3 Biologicals C myc Cell Cyclophilin b Erk1 Glut 1 Grp78 Menin Novus Raptor Tfeb Tsc1 Tsc2 Tubulin

Corresponding Organization :

Other organizations : The University of Texas Southwestern Medical Center, Broad Institute

Top 5 similar protocols

Protocol cited in 6 other protocols

Variable analysis

independent variables

Cell lysates and western blotting was performed as previously described (Vega-Rubin-de-Celis et al, 2010 (link))
Antibodies from the following sources: S6K1, phospho-S6K1 (T₃₈₉), S6, phospho-S6 (S_235/236), total 4E-BP1, phospho-4E-BP1 (T_37/46), phospho-4E-BP1 (S₆₅) from Cell Signaling; HIF-1α, TSC1 and Menin from Bethyl laboratories; TSC2, ATF4 and c-Myc from Santa Cruz; Raptor from Millipore; Erk1/2, phospho-Erk1/2 (T₁₈₃/Y₁₈₅) and Tubulin from Sigma; Cyclophilin B from Abcam; GLUT-1 from Novus Biologicals; and Grp78 from BD Biosciences
Atp6v1a and Atp6v1b2 antibodies were a generous gift from D Brown (Massachusetts General Hospital, Boston, MA)
Atp6v0a1 and Atp6v0a3 antibodies were kindly provided by V Marshansky (Massachusetts General Hospital, Boston, MA)
Tfeb antibodies were developed in collaboration with Bethyl Laboratories

dependent variables

Protein expression and phosphorylation levels measured by western blotting

control variables

Tubulin
Cyclophilin B

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!