Fig. 5
From: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis
SUMO1 modification of KHSRP promotes its cytoplasmic localization. a K87R mutation affects the localization of HA-KHSRP. HeLa cells were transfected with HA-KHSRP-WT, HA-KHSRP-K87R or HA-KHSRP-ΔNLS, respectively. Nuclear and cytosolic fractions were extracted by the Nuclear/Cytosol fractionation kit. Nuclear and cytoplasmic fraction extracts were immunoblotted with indicated antibodies. b SUMO1 modification affects the localization of HA-KHSRP by using the method of immunofluorescent staining. HeLa cells transfected with HA-KHSRP-WT or HA-KHSRP-K87R with GFP-SUMO1 were stained with the primary antibody anti-HA (Rabbit), and then with the second antibody of Alexa Fluor 568 anti-rabbit. DAPI staining was to visualize the nucleus. The green signals indicated the expression of GFP-SUMO1 carrying Green Fluorescent Protein and the images of GFP-SUMO1 were directly taken without staining. All the images were taken by Nikon microscope. Scale bar, 25 μm. c-d SUMO1 modification of KHSRP promotes its cytosolic localization. Flag-SUMO1-KHSRPΔN was constructed by replacing the N-terminal (aa 1–67) of KHSRP with Flag-tagged SUMO1(aa 2–96). c HeLa cells transfected with Flag-KHSRPΔN or Flag-SUMO1-KHSRPΔN were extracted by the Nuclear/Cytosol fractionation kit, and followed by immunoblotting with indicated antibodies. d HeLa cells transfected with Flag-KHSRPΔN, Flag-SUMO1-KHSRPΔN or Flag-KHSRPΔNLS were stained using the primary antibody of anti-Flag M2 and the secondary antibody of Alexa Fluor 488 (anti-mouse). e-f SUMOylation increases the cytosolic localization of endogenous KHSRP. e HeLa-shControl, HeLa-shSENP1 and HeLa-shUbc9 cells were extracted by the Nuclear/Cytosol fractionation kit, then immunoblotted with indicated antibodies. f These three cell lines were stained using the primary antibody anti-KHSRP (Rabbit) and the secondary antibody Alexa Fluor 488 (anti-rabbit). Images were taken by Nikon microscope, and the cytoplasmic location of KHSRP was indicated by red arrows. The same scale bar (25 μm) was used in all images (b, d, f). The staining cells numbers were counted with the Image J software and the statistical analysis was performed (b, d, f). The band intensities were calculated by Image J and quantified by normalization to GAPDH and LMNB1, the percentage of nuclear/cytosolic fraction of every sample was calculated (a, c, e)