Western Blots analysis in KEP rabbit cells
Description
Original western blot images of rabbit iPS cell lines (KEP cell lines) in 4 experiments: - Analysis of AKT and Ph-AKT expression in B19_KF and KEP#37_KF cells (AKT KEPc 37 figS3 and pAKT KEPc 37 figS3). - Expression of KLF2-V5 and HA-ERAS in KEPi#13, KEPi#18 before and after withdrawal of Shield1 (KEPi 13 and 18 HA and V5 Fig6). - Expression of KLF2-V5 and HA-ERAS in KEPi#28, and KEPi#36 cells before and after withdrawal of Shield1 (KEPi 28 and 36 HA and V5 Fig6). - Analysis of AKT and Ph-AKT expression in B19, KEPc#13_VAL+S, KEPc#13_VAL+S-S, KEPc#18_VAL+S, and KEPc#18_VAL+S-S cells (AKT KEPi 13 18 28 36 figS6 and pAKT KEPi 13 18 28 36 figS6).
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Cells were lysed in cold RIPA lysis buffer (0.5% NP-40, 1% Triton-X, 10% glycerol, 20 mM Hepes pH 7.4, 100 mM NaCl, 1 mM sodium orthovanadate, 0.1% DTT, and protease inhibitors (Roche, #05 892 970 001) for four hours at 4°C. Lysates were cleared by centrifugation for 15 min and stored at -80oC. Protein concentrations were measured using Bradford assay. For SDS-PAGE electrophoresis, 30µg of total proteins were loaded onto each well of Mini-PROTEAN TGX Stain-Free Precast Gels (10%, Biorad, # 4568031), and migrated for 45 min at 120 Volts. Precision Plus Protein Dual Color Standards (Biorad, # 1610374) were used as protein ladder. After electroporation, proteins were transferred onto membranes using Trans-Blot® TurboTM RTA Midi 0.2 µm Nitrocellulose Transfer Kit (Biorad, #1704271). The membranes were subsequently blocked in TBST solution (200 mM Tris-HCl, 1.5 M NaCl, 0.1% Tween-20, 5% milk) for one hour at room temperature, prior to incubation with primary antibodies diluted in TBST at 4°C for 12 hours. Primary antibodies include: anti-V5 Tag (Invitrogen, ref R96025, 1:500, anti-HA (Sigma-Aldrich, ref H6908, 1:500), anti-Akt (pan) (Cell Signaling, ref #4691, 1:1000), anti-Phospho-Akt (Ser473) (Cell Signaling, ref #4060, 1:1000), and anti-beta-actin (Sigma-Aldrich, ref A3854, 1:10000). Membranes were incubated with HRP-conjugated secondary antibody (Jackon ImmunoResearch anti-mouse ref 211-032-171 and anti-goat ref 115-035-146, dilution 1:5000) for one h at room temperature. After serial washing in TBST, HRP activity was revealed using ClarityTM Western ECL substrate (Biorad, #170-5060) and ChemiDocTM MP imaging system (Biorad).