Immunoblot Analysis of Protein Expression

Whole-cell lysates were homogenized in modified RIPA buffer^{14 (link)} and assayed by immunoblot as previously described^{14 (link)}. The primary antibodies against FKBP51 (rabbit polyclonal; Novus Biological), FKBP51s^{13 (link)} and CD274/PD-L1 (rabbit polyclonal; Novus Biological) were used diluted 1:2500. CD133 (rabbit polyclonal; Abcam; Cambridge, UK) was used diluted 1:1000. A further antibody Pdcd-1L1 (rabbit polyclonal, Santa Cruz Biotechnology; Santa Cruz, CA, USA) was used for PD-L1 detection at the 1:1000 dilution. Antibody against M2-Flag, caspase-7 and γ-Tubulin (mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA) were used diluted 1:5000. Anti β-Actin-Peroxidase (mouse monoclonal; Sigma-Adrich) was used diluted 1:10000. Anti-phospho-Akt (Ser473), Akt1/2/3, phosphor-S6 kinase, G3PDH and Sox-2 (rabbit monoclonal; Cell Signaling, Danvers, MA, USA) were used diluted 1:1000. Anti-p70S6 kinase (rabbit polyclonal; Santa Cruz) was used diluted 1:500.

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D’Arrigo P., Digregorio M., Romano S., Tufano M., Rea A., Hausch F., Dedobbeleer M., Vigorito V., Russo S., Bauder M., Rogister B, & Romano M.F. (2019). The splicing FK506-binding protein-51 isoform plays a role in glioblastoma resistance through programmed cell death ligand-1 expression regulation. Cell Death Discovery, 5, 137.

Publication 2019

CD133 Anti β-Actin-Peroxidase Anti-p70S6 kinase

Actin Akt1 Antibodies Antibody Biological Caspase 7 Cell Dilution Fkbp51 Mouse Novus P70s6 kinase Pd l1 Peroxidase Phosphor Rabbit Ripa S6 kinase Sox 2 Tubulin

Corresponding Organization :

Other organizations : University of Naples Federico II, University of Liège

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Variable analysis

independent variables

Treatment with primary antibodies against FKBP51, FKBP51s, CD274/PD-L1, CD133, Pdcd-1L1, M2-Flag, caspase-7, γ-Tubulin, β-Actin-Peroxidase, phospho-Akt (Ser473), Akt1/2/3, phosphor-S6 kinase, G3PDH, Sox-2, and p70S6 kinase

dependent variables

Protein levels of FKBP51, FKBP51s, CD274/PD-L1, CD133, Pdcd-1L1, M2-Flag, caspase-7, γ-Tubulin, β-Actin, phospho-Akt (Ser473), Akt1/2/3, phosphor-S6 kinase, G3PDH, Sox-2, and p70S6 kinase

control variables

Modified RIPA buffer used for cell lysis
Immunoblot analysis as previously described

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