Fkbp12

MV4;11 and 293T cells were lysed with RIPA buffer supplemented with halt protease inhibitor cocktail (Thermo Fisher Scientific) and 0.1% benzonase (Novagen) on ice for 60 minutes. Lysates were clarified by centrifugation at 20,000 xg for 10 minutes at 4 ºC. NIH/3T3 and 293FT cells were lysed with RIPA buffer supplemented with cOmplete protease inhibitors (Roche) and PhosSTOP phosphatase inhibitors (Roche) on ice for 60 minutes. Lysates were clarified by centrifugation at 20,000 xg for 10 minutes at 4 ºC. Protein concentration was determined by a BCA assay (Pierce), and all samples were run with equal total protein content. The following antibodies were used in this study: HA (Cell Signaling, #3724 and #2367), BRD4 long isoform (Bethyl labs, #A301-985A), BRD4 short isoform (Abcam, #ab128874), BRD3 (Abcam, #ab56342), BRD2 (Bethyl labs, #A302–582A), FKBP12 (Abcam, #ab24373), vinculin (Santa Cruz, #Sc-25336), GAPDH (Millipore, #MAB374), phospho-AKT S473 (Cell Signaling, #4060), AKT (Cell Signaling, #2920), phospho-MEK1/2 S221 (Cell Signaling, #2338), MEK1/2 (Cell Signaling, #4694), phospho-ERK1/2 T202/Y204 (Cell Signaling, #4370), ERK1/2 (Cell Signaling, #4696), phospho-Pol II S2 (Millipore, #04-1571-1), and Pol II (Santa Cruz, #Sc-899). Imaging was performed by detection of fluorescently labelled infrared secondary antibodies (IRDye) on the Odyssey CLx Imager (LI-COR).

Protein pellets (isolated as described for the DNA extraction) were suspended in 5x pellet volume of RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5 % sodium deoxycholate, 0.1 % SDS, 1 % triton X-100) and incubated on ice for ~30 min. The lysate was spun for 10 min at 4oC, at 14,000 rpm and the supernatant was combined with 4x SDS gel-loading buffer (62.5 mM Tris-H₃PO₄, pH 7.5, 1 mM EDTA, 2 % SDS, 10 mM DTT, 1 mM NaN₃ and 33% glycerol) and fresh 15% β-mercaptoethanol. Samples were boiled at 100oC for 5 min and loaded on SDS-PAGE gradient gel (Biorad). Electrophoresis was performed using Mini-PROTEAN Tetra cell electrophoresis chamber (Qiagen) for 2 h at 120 V. The resolved proteins were transferred on Whatman nitrocellulose membrane using a trans-blot electrophoretic transfer system according to the manufacturer’s protocol, blocked with 5% milk PBST and probed with the antibodies against FKBP-12 (1:500), Cre (1:500) and enolase (1:1000) (all Abcam), followed by a compatible HRP-labelled secondary antibody. Enhanced chemiluminescence system (ECL) was used to visualise the proteins on X-ray film.