Activation of p38 MAPK and MK2 is required for efficient nuclear translocation of SRC-3 in response to TNF-α. ( A , B ) p38 MAPK and MK2 is involved in nuclear translocation of SRC-3. A549 WT cells were seeded on coverslip and left overnight. The next day, cells were treated with either DMSO, 10 ng/ml TNF-α or <t>SB-202190</t> ( A ) or 10 μM PF-3644022 ( B ) separately, or in combination with SB-202190 ( A ) or PF-3644022 ( B ) for 30 min followed by TNF-α stimulation for 60 min. Representative images of the SRC-3 WT A549 cells stained for SRC-3 (red) using anti-SRC-3 antibody and nucleus ( blue, DAPI). The specificity of the antibody for SRC-3 was verified using SRC-3 KO cells (Supplementary Fig. S2 A,B). ( C ) Generation of SRC-3 KO A549 cells. Expression of SRC-3 and actin in SRC-3 WT and SRC-3 KO A549 cells were analyzed by Western-blotting. ( D ) SRC-3 WT is more efficiently translocated into nucleus than SRC-3 S857A in response to TNF-α. SRC-3 KO A549 cells were seeded in 24 well plate and left overnight. The next day, the cells were transfected with 200 ng of vector expressing either SRC-3 wild type (WT)-FLAG or SRC-3 S857A-FLAG. After 48 h, the cells were either stimulated with 10 ng/ml TNF-α for 60 min or left unstimulated. Representative images of the SRC-3 KO A549 cells stained for SRC-3 (red, anti-SRC-3) and nucleus (blue, DAPI). ( E – G ) Quantitative presentation of the distribution of SRC-3 in conditions described in ( A , B , D ) respectively. The cellular localization of SRC-3 was determined as either cytoplasmic and nuclear or mainly nuclear. The SRC-3 overlapping nucleus (DAPI) is considered nuclear and the SRC-3 overlapping the nucleus and present around and outside the nucleus is considered cytoplasmic + nuclear. For quantification, minimum 100 cells were counted for each condition described in ( A , B , D ) and expressed in percentage. Data in ( E , F , G ) are presented as mean ± SD of three replicates. Unpaired t-test was used for analysis of significance between groups compared in the figure. * P