Western Blot Analysis of Signaling Proteins

Cells were lysed in 1% NP-40 lysis buffer as previously described (Delgoffe et al., 2013 (link)). Lysates were separated by SDS-PAGE on 4–12% Bolt or Bio-Rad gels, transferred to polyvinylidene difluoride membranes, and blocked in 3% BSA in Tris-buffered saline with 0.1% Tween-20. Primary antibodies to phospho-p38-MAPK, phospho-ATF2, Mitofusin-2, and actin (Cell Signaling Technologies) were added in 3% BSA/TBST for 1–2 h at room temperature or overnight at 4°C. Secondary antibodies (anti-rabbit or anti-mouse linked to horseradish peroxidase; Jackson ImmunoResearch) were added in 3% BSA/TBST for 1–2 h at room temperature. Enhanced chemiluminescence was visualized by using Western Lightning (PerkinElmer).

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Menk A.V., Scharping N.E., Rivadeneira D.B., Calderon M.J., Watson M.J., Dunstane D., Watkins S.C, & Delgoffe G.M. (2018). 4-1BB costimulation induces T cell mitochondrial function and biogenesis enabling cancer immunotherapeutic responses. The Journal of Experimental Medicine, 215(4), 1091-1100.

Publication 2018

phospho-p38-MAPK phospho-ATF2 Mitofusin-2 Western Lightning

Actin Antibodies Atf2 Buffer Cells Chemiluminescence Gels Horseradish peroxidase Membranes Mitofusin 2 Mouse Np 40 P38 mapk Polyvinylidene difluoride Rabbit Saline Sds page Tween 20

Corresponding Organization : University of Pittsburgh

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Variable analysis

independent variables

1% NP-40 lysis buffer

dependent variables

Phosphorylation of p38-MAPK
Phosphorylation of ATF2
Mitofusin-2 expression
Actin expression (as a control)

control variables

SDS-PAGE on 4–12% Bolt or Bio-Rad gels
Polyvinylidene difluoride membranes
3% BSA in Tris-buffered saline with 0.1% Tween-20 for blocking
Primary antibodies to phospho-p38-MAPK, phospho-ATF2, Mitofusin-2, and actin (Cell Signaling Technologies)
Secondary antibodies (anti-rabbit or anti-mouse linked to horseradish peroxidase; Jackson ImmunoResearch)
Enhanced chemiluminescence visualization using Western Lightning (PerkinElmer)

controls

Actin as a loading control

Annotations

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