Cell were seeded in 6-well dishes at 30,000 cell/well and induced with
doxycycline for to 18 h prior to harvesting. Cell lysates were prepared in 1x
sample buffer (160 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.05% bromophenol
blue). Equal amounts of cell lysates were separated by a 10% SDS-PAGE and
transferred onto PVDF membrane. GFP purification samples for immunoblotting were
collected by elution with sample buffer. The membrane was developed with the
appropriate antibodies and ECL. Immunoblots were analyzed using ChemiDoc imaging
system (BioRad). The images were subjected to linear contrast/brightness
enhancement in Photoshop (CS6, 13.0.6 x64, extended) when needed for data
representation purposes. Antibodies used in the study include: GFP (JL-8,
Clontech), TCP1 (91A, ThermoFisher), CCT2 (kind gift from Willianne Vonk/Judith
Frydman), GAPDH (clone 6C5, mAb374, Millipore), Penta His (Qiagen, Hilden,
Germany), TAF5 (in-house, 25TA 2G7) and TAF6 (in-house, 1TA-1C2).
doxycycline for to 18 h prior to harvesting. Cell lysates were prepared in 1x
sample buffer (160 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 0.05% bromophenol
blue). Equal amounts of cell lysates were separated by a 10% SDS-PAGE and
transferred onto PVDF membrane. GFP purification samples for immunoblotting were
collected by elution with sample buffer. The membrane was developed with the
appropriate antibodies and ECL. Immunoblots were analyzed using ChemiDoc imaging
system (BioRad). The images were subjected to linear contrast/brightness
enhancement in Photoshop (CS6, 13.0.6 x64, extended) when needed for data
representation purposes. Antibodies used in the study include: GFP (JL-8,
Clontech), TCP1 (91A, ThermoFisher), CCT2 (kind gift from Willianne Vonk/Judith
Frydman), GAPDH (clone 6C5, mAb374, Millipore), Penta His (Qiagen, Hilden,
Germany), TAF5 (in-house, 25TA 2G7) and TAF6 (in-house, 1TA-1C2).