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. 2016 Feb 12;7(11):12975–12996. doi: 10.18632/oncotarget.7349

Figure 3. Proteomic and cell biology analyses of AR-12 interacting proteins.

Figure 3

(A) As described in the Methods, AR-12 conjugated via biotin to sepharose beads was used to capture proteins from a whole cell lysate and those proteins specifically associating with beads in an AR-12–dependent fashion were determined after proteolytic digestion in a mass spectrometer. (B) GBM5 and GBM12 cells were treated with vehicle, OSU-03012 (2.0 μM) / sorafenib (2.0 μM) and/or sildenafil (2 μM) for 6 h after which: (a) in 96 well plates, cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the expression level of HSP27 presented at 10X and 60X magnification; (b) cells were lysed with bromophenol blue buffer and subjected to SDS PAGE followed by immuno-blotting to detect the expression level of HSP27. (C) GBM5, GBM6 and GBM12 cells were treated with vehicle, OSU-03012 (0–3.0 μM) or sorafenib (0–3.0 μM) for 3 h after which cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the expression level of HSP27. The relative fluorescence intensity value from 40 different cells from each condition was determined using Hermes system software (+/− SEM). (D) GBM12 cells were treated with vehicle, OSU-03012 (2.0 μM) / sorafenib (2 μM) and/or sildenafil (2 μM) for 2 h after which cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the expression level of HSP27, using antibodies that recognize epitopes in the NH2-terminus; COOH-terminus; and in the middle of the protein, as well as our previously used “usual” antibody. (E) GBM5 and GBM12 cells were treated with vehicle, OSU-03012 (2.0 μM) and/or sildenafil (2 μM) for 6 h after which cells were fixed in place and permeabilized using 0.5% Triton X100. Immuno-fluorescence was performed to detect the total expression level of AKT and p38 MAPK, and the phosphorylation levels of AKT T308; p38 MAPK; mTOR S2448; HSP27 S15; HSP27 S78; and HSP27 S82.